Molecular mechanisms involved in the response to desiccation stress and persistence in Acinetobacter baumannii.

Article pubs.acs.org/jpr

Molecular Mechanisms Involved in the Response to Desiccation Stress and Persistence in Acinetobacter baumannii Carmen M. Gayoso,†,§ Jesús Mateos,‡ José A. Méndez,† Patricia Fernández-Puente,‡ Carlos Rumbo,† María Tomás,† Ó skar Martínez de Ilarduya,‡,§ and Germán Bou*,† †

Microbiology Division, INIBIC-Complejo Hospitalario Universitario de la Coruña, Xubias s/n, 3a Planta Ed. Sur 15006 La Coruña, Spain ‡ Rheumatology Division, Proteored-ISCIII INIBIC-Complejo Hospitalario Universitario de la Coruña, Xubias s/n, 3a Planta Ed. Sur, 15006 La Coruña, Spain S Supporting Information *

ABSTRACT: Desiccation tolerance contributes to the maintenance of bacterial populations in hospital settings and may partly explain its propensity to cause outbreaks. Identification and relative quantitation of proteins involved in bacterial desiccation tolerance was made using label-free quantitation and iTRAQ labeling. Under desiccating conditions, the population of the Acinetobacter baumannii clinical strain AbH12O-A2 decreased in the first week, and thereafter, a stable population of 0.5% of the original population was maintained. Using label-free quantitation and iTRAQ labeling, 727 and 765 proteins, respectively, were detected; 584 of them by both methods. Proteins overexpressed under desiccation included membrane and periplasmic proteins. Proteins associated with antimicrobial resistance, efflux pumps, and quorum quenching were overexpressed in the samples subjected to desiccation stress. Electron microscopy revealed clear morphological differences between desiccated and control bacteria. We conclude that A. baumannii is able to survive long periods of desiccation through the presence of cells in a dormant state, via mechanisms affecting control of cell cycling, DNA coiling, transcriptional and translational regulation, protein stabilization, antimicrobial resistance, and toxin synthesis, and that a few surviving cells embedded in a biofilm matrix are able to resume growth and restore the original population in appropriate environmental conditions following a “bust-and-boom” strategy. KEYWORDS: proteome, desiccation, persistence, dormancy, Acinetobacter baumannii, Cpx pathway

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INTRODUCTION

A. baumannii can cause prolonged infections and can also persist via asymptomatic colonization of patients, which enables its spread both within and between hospitals. In summary, both desiccation tolerance and multidrug resistance may contribute to the maintenance of the species in the hospital setting and may partly explain its propensity to cause prolonged outbreaks of nosocomial infections.5 Between January 2006 and May 2008, a multiresistant epidemic clone was obtained from 290 patients of Hospital 12 de Octubre (Madrid) harboring A. baumannii antibiotype 1 (clone AbH12O-A2); 165 of the patients were infected (57%), and 125 (43%) were colonized.6 Clone AbH12O-A2 had unique characteristics. First, it was a multi-drug-resistant clone, susceptible only to tigecycline and colistin. Second, it harbored a carbapenemase blaOXA‑24 gene, flanked by XerC/XerD binding sites located on a plasmid. Third, this clone

The acquisition of infections in hospital settings is a growing problem in Intensive Care Units (ICUs). Since 1980, experts in infectious disease have recognized that patients in ICUs acquire nosocomial infections at a rate 5−10 times higher than in other hospital settings.1,2 Acinetobacter spp. are ubiquitous Gram-negative bacteria which can cause a wide range of opportunistic infections, mainly in elderly patients, infants, and patients with severe underlying disease.3 Patients in ICUs, who often require assisted mechanical ventilation and urinary or intravascular catheters, are at particular risk. Acinetobacter baumannii is an emerging human pathogen and a significant cause of nosocomial infections among hospital patients worldwide. Other factors in addition to drug resistance have greatly contributed to the emergence of A. baumannii as a significant hospital pathogen. Such factors include the resistance to desiccation, which enables the species to withstand dry environments for months,4 thus facilitating its spread via hospital personnel, infrastructure, and medical devices. Finally, © XXXX American Chemical Society

Received: June 24, 2013

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dx.doi.org/10.1021/pr400603f | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

overexpressed two putative virulence factors, septicolysin and TonB-dependent receptor. Although most studies focus on bacteria growing in nearoptimal conditions, slow replication is the norm in the microbial world. Virtually all microbial species require the ability to withstand changing environments or periods when basic requirements for growth are missing, and it is a known fact that the most common nosocomial pathogens may survive or persist on surfaces for months and can be a continuous source of transmission.7 In general, three basic strategies can be outlined: bust and boom, cellular quiescence, and true dormancy (recently reviewed in Rittershaus et al.8). In the bust-and-boom strategy, a few surviving cells are able to resume growth and replicate rapidly once the environmental conditions are suitable. Cellular quiescence refers to an ability of the bulk of the bacterial population, when exposed to growth-limiting stress, to slow or arrest its growth in order to persist in a viable nonreplicating state while still displaying metabolic capacity and maintaining their membrane potential without undergoing obvious morphological differentiation. In a true dormancy, proper sporulation takes place, resulting in hardy metabolically inactive cells. However, still relatively little is known about the regulatory mechanisms and physiological changes that define microbial quiescence. The present study was aimed at the identification of the mechanisms that enable A. baumanni clinical strain AbH12OA2 to survive extreme conditions (lack of nutrients and water), such as those often encountered in hospital settings, and at the characterization of the response to stress by desiccation. Two proteomics approaches, label-free quantitation and relative quantitation using iTRAQ reagents, were used to compare the proteome of A. baumannii subjected to desiccation stress (relative humidity 30% for 30 days) with that of a control culture of this strain (OD = 0.8). The present study is the first experimental approach aimed at identifying protein determinants of persistence in a nosocomial bacterial pathogen.

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Figure 1. Acinetobacter baumannii cells were plated on 6-well culture plates and kept at a 21 °C, 30% RH for 30 days. Proportion of surviving cells along time, measured as a percentage of the original number of colony-forming units (CFU), is shown.

Protein Extraction

A. baumannii planktonic cells were harvested by centrifugation (3500g, 10 min, 4 °C) and washed twice with 0.9% (w/v) NaCl. The protein lysates of planktonic/desiccated cells were obtained by mechanical disruption. Briefly, total protein fractions obtained from pellets were thawed in 3 or 5 mL of lysis buffer [65 mM NaH2PO4, 50 mM Na2HPO4, 1 mM MgSO4·7H2O, 30 or 50 μL of protease inhibitor mix (GE Healthcare, Piscataway, NJ) and 30 or 50 μL of nuclease mix (GE Healthcare), depending on pellet mass] and sonicated for 3 × 5 min (0.5 cycle, 60 amplitude, 4 °C). Unbroken cells and cell debris were removed by centrifugation (1500g for 10 min and 3600g for 30 min, at 4 °C), and the supernatants were clarified through a 0.45 μm filter (Millipore, Billerica, MA). The concentration of protein was measured using the Bio-Rad protein assay (Bio-Rad, Munich, Germany). For gel-based analyses, the protein extracts were processed with a 2D clean-up kit (GE Healthcare), following the manufacturer’s instructions. Samples for label-free quantitation were resuspended in 8 M urea.

EXPERIMENTAL SECTION

Culture of the Clinical Strain

Label-Free Mass Spectrometry Analysis and Relative Quantitation

A. baumannii strain AbH12O-A2 was grown in 500 mL of Mueller−Hinton liquid medium. Once the OD of the culture reached 0.8−1, the cells were precipitated, washed with normal saline, and resuspended in 10 mL of normal saline. Aliquots (300 μL) of this suspension were deposited on adherent 6-well culture plates. The plates were maintained at 21 °C and at a constant low relative humidity (RH) of 30%, by placing them in sealed boxes containing silica. The temperature and humidity in the boxes were monitored with thermohygrometers.

Label-free quantitation by LC-MALDI-TOF/TOF analysis was performed as previously described.10 Briefly, 40 μg of protein for each condition (three control and three desiccated samples) was size-fractionated by 1D SDS-PAGE (10% acrylamide), and the entire lane was divided in 12 sections, which were processed individually. After standard trypsin digestion and peptide extraction, peptides were desalted using homemade Stage Tips11 and separated using reversed-phase chromatography in a nanoLC system (Tempo, ABSciex). Peptides were desalted and concentrated in a trapping column (0.5 × 2 mm, MichromBioresources, Auburn, CA) at a flow rate of 15 μL/ min for 15 min and loaded onto a C18 column (Integrafit C18, Proteopep II, 75 μm i.d., 15 cm, 5 μm, 300 Å; New Objective, Woburn, MA). Elution of peptides was done at a flow rate of 0.35 μL/min during a 1 h linear gradient from 2 to 40% B (mobile phase A: 0.1% trifluoroacetic acid, 2% acetonitrile; mobile phase B: 0.1% trifluoroacetic acid, 95% acetonitrile), mixed with α-cyano matrix (4 mg/mL at a flow rate of 1.2 μL/ min), automatically deposited on a MALDI plate using a MALDI spotter (SunCollect, Sunchrome, Friedrichsdorf, Germany), and analyzed in a 4800 TOF/TOF system

Growth Curve

Samples of the bacteria were removed after 0, 3, 5, 7, 9, 15, 21, and 30 days. Cells were scraped from the culture plates and recovered in normal saline. Different dilutions were plated on Mueller−Hinton, and the number of colonies was counted after 1 day at 37 °C (Figure 1). Biofilm Analysis by Crystal Violet Staining

Control and desiccation-stressed samples were stained with crystal violet, as described in Djordjevic et al.9 Since the values of stress desiccation samples were out of range, measurements of a 1:4 dilution in acetone were made. B



Article

(ABSciex). The chromatograms were composed by 240 spots, each one comprising a 15 s deposition. The 4000 series Explorer v.4.2 (ABSciex) software was used to generate both the spectra and peak list. Plate model and default calibration of the MALDI plate were done with a laser voltage of 3400 kV and 1000 shots/spectrum. Samples were automatically analyzed in MS mode with a laser voltage of 3800 kV and 1500 shots/ spectrum. Automated precursor selection was carried out using a Jobwide interpretation method, which selects up to 12 precursors per fraction. The signal to noise lower threshold (50) and trypsin autolytic peptides and other background ions were excluded. The laser voltage was 4800 kV, and 2000 shots/ spectrum were acquired using a medium range CID collision energy. LC-MALDI-TOF/TOF data were analyzed using Protein Pilot 4.0 software (ABSciex) as search engine for protein identification. Protein Pilot search parameters were as follows: Cys-alkylation, iodoacetamide; ID focus, biological modifications; digestion, trypsin; database, last SwissProt release; species filtering, none; search effort, thorough ID and detection protein threshold unused ProtScore (Conf > 1.3 (95.0%)). Scoring model was defined by the Paragon algorithm. False discovery rate (FDR) was estimated in less than 1% by doing the searching in parallel against a decoy database using “PSPEP on” mode (data not shown). For manual relative quantification, the number of Protein Pilot unique peptides identified with a confidence greater than 95% for a given protein in the different experimental replicates (three controls vs three desiccations) was used to calculate up to nine individual ratios of control/desiccation representatives of the relative abundance of the protein that were subsequently used to calculate an average ratio. A Mann−Whitney statistical test was applied considering the number of peptides found in the control and desiccation samples. Proteins showing differential expression at a P value 1. The first two principal components accounted for 92.7% of the total variability, resulting in a clear separation of the control and desiccation-stressed samples (Figure 1 in Supporting Information). On the basis of the components loadings, the principal components were

Proteomics Approach

Proteins extracted from control or desiccation-stressed samples (bacteria kept for 30 days at 21 °C, 30% RH) were analyzed by iTRAQ or label-free quantitation methods. Proteins were identified by comparison with a database containing all A. baumannii strains sequenced so far since strain AbH12O-A2 has not been sequenced and any comparison against a single strain would result in loss of information. A comparison was made with the AB0057 strain in order to determine the number of proteins identified by each approach and to check which ones coincided in both techniques. The results showed that 727 proteins were detected by label-free quantitation and 765 proteins were detected by iTRAQ, with 584 of the proteins being detected by both approaches (Figure 2). The number of D



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Table 1. Proteins Detected by Label-Free Quantitation (P < 0.05)a

protein name

Uniprot accession number

strain (Supporting Information Table 3)

control

desiccation

Nr peptides/sample (coverage 95%)


Upregulated proteins detected by the label-free quantitation technique in desiccation-stressed samples Transcription integration host factor subunit alpha D6JW35 ACIG3 2/2/2 (20.40−20.41) Translation ribosome-recycling factor D0C6G8 ACIBA 4/2/1 (25.54) 50S ribosomal protein L18 D6JXA7 ACIG3 0/0/0 Proteins associated with antimicrobial resistance β-lactamase AmpC Q7WZ37 ACIG3 59/53/56 (58.56−62.14) carbapenem-associated resistance protein (CarO) C0L0K3 ACIBA 4/4/5 (30.08−31.30) putative outer membrane protein (Omp25) Q4A208 ACIBA 2/3/1 (5.15−10.30) AdeI Q2FD95 ACIBA 2/1/3 (3.36−13.22) AdeK Q2FD92 ACIBA 5/2/4 (0.00−18.76) β-lactamase Oxa51 Q5QT35 ACIBA 0/0/0 TolA D0CEI7 ACIBA 0/0/0 putative RND type efflux pump involved in aminoglycoside D6JYT9 ACIG3 4/3/5 (13.73−19.70) resistance (AdeT) Other outer membrane proteins putative uncharacterized protein (putative DcaP-like) B7I6Y2 ACIB5 2/2/4 (4.15−8.99) outer membrane lipoprotein Blc D0CBS0 ACIBA 0/0/0 porin D D0CB85 ACIBA 0/0/0 ROS RNS detoxification catalase HPII (hydroxyperoxidase II) D0C8B2 ACIBA 17/11/31 (20.22−41.57) superoxide dismutase [Cu−Zn] D0CCT4 ACIBA 6/5/3 (25.48−25.48) catalase Q83WC7 ACICA 0/0/0 glutathione peroxidase B7I1W7 ACIBA 0/0/0 Chaperones ClpB. ATP-dependent protease, Hsp 100, part of D0CEH1 ACIBA 4/6/6 (8.03−11.06) multichaperone system with DnaK, DnaJ, and GrpE Uncharacterized proteins putative uncharacterized protein D0CFS8 ACIB5 3/3/2 (19.29) putative uncharacterized protein D0C5R3 ACIBA 0/0/0 Adherence and motility type VI secretion system OmpA/MotB D0C9R5 ACIBA 8/9/6 (37.79−65.44) CsuC B7IC62 ACIB5 2/2/2 (8.30−9.52) Proteins related to quorum quenching metallo-β-lactamase superfamily protein; putative LB2HXB4 ACIBA 0/0/0 ascorbate 6-phosphate lactonase Metabolism alcohol dehydrogenase D6JTQ2 ACIG3 0/0/0 aspartate/tyrosine/aromatic aminotransferase B2HUD5 ACIBC 17/12/18 (24.25−41.92) predicted hydrolase or acyltransferase (α/β hydrolase B2I233 ACIBC 0/0/0 superfamily) thiol:disulfide interchange protein D6JYI2 ACIG3 6/4/3 (22.44−36.59) glutamate/aspartate transporter D0C807 ACIBA 2/2/2 (5.39−5.39) putative uncharacterized protein (EsvE2) D6JUG6 ACIG3 0/0/0 glutaminase-asparaginase (L-asparagine/LD0C833 ACIBA 4/2/3 (3.66−16.62) glutamineamidohydrolase) Secreted proteins putative 17 kDa surface antigen B2HX32 ACIBC 0/0/0 LysM domain-containing protein D0C9K9 ACIBA 0/0/0 LemA family protein D0CCP5 ACIBA 8/7/5 (43.88−60.71) YceI family protein D0C7 V7 ACIBA 3/2/2 (14.29) rhodanese domain protein D0CD68 ACIBA 0/0/0 MviM protein D0CEV6 ACIBA 0/0/0 conidiation-specific protein 10 family protein B7I3G3 ACIB5 0/0/0 WecC protein A3M0 V3 ACIBT 0/0/0 Proteins associated with quorum quenching dihydrocoumarin hydrolase Q83WC8 ACICA 0/0/0

E

3/4/4 (35.00−23.47) 17/10/21 (63.69) 2/2/3 (11.21−27.23) 89/84/87 (59.79−65.23) 11/33/15 (41.77−52.61) 5/7/4 (22.31−29.18) 17/17/13 (39.90−47.11) 13/5/12 (18.76−33.40) 2/4/3 (8.76−12.00) 4/8/7 (64.04) 5/9/9 (18.21−37.90)

7/4/5 (14.75−19.59) 7/2/5 (39.50−45.64) 2/2/2 (3.88−14.80) 59/65/35 (43.29−46.70) 9/11/10 (25.48−58.17) 2/2/2 (6.52) 2/3/2 (18.08−33.15) 15/15/27 (23.52−34.23)

15/12/17 (30.01−64.97) 4/2/3 (33.77) 12/17/14 (65.44−71.40) 4/8/5 (17.95−30.40) 2/2/2 (9.98−10.07)

2/2/2 (2.77−7.70) 47/20/29 (43.80−60.34) 8/14/11 (33.19−65.11) 5/14/12 (33.17−62.93) 7/8/13 (8.42−19.53) 2/3/2 (13.89−12.00) 6/5/5 (16.62−17.75)

3/8/5 (21.05−22.97) 2/8/5 (12.96−63.69) 11/17/8 (60.71−80.10) 4/7/5 (30.60−36.73) 2/2/2 (11.45−39.42) 2/2/2 (7.28−18.70) 2/3/2 (5.72−16.24−14.40) 5/2/2 (17.63−17.20) 6/6/7 (18.48−38.77)



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Table 1. continued

protein name



control

desiccation



Upregulated proteins detected by the label-free quantitation technique in desiccation-stressed samples Toxins hemolysin septicolysin

D0C8L9 ACIB5 0/0/0 3/4/8 (28.84−40.02) B7ICI9 ACIBA 0/0/0 3/2/2 (14.58−25.69) Downregulated proteins detected by the label-free quantitation technique in desiccation-stressed samples

Transcription elongation factor Tu translation elongation factor Ts DNA-directed RNA polymerase subunit alpha (RpoA) DNA-directed RNA polymerase (RpoC) elongation factor P elongation factor G transcription termination factor NusA translation initiation factor IF-2 transcriptional accessory protein Translation ribosomal protein S1 50S ribosomal protein L3 30S ribosomal protein S7 50S ribosomal protein L9 50S ribosomal protein L14 50S ribosomal protein L15 50S ribosomal protein L31 30S ribosomal protein S8 30S ribosomal protein S16 30S ribosomal protein S5 50S ribosomal protein L27 prolyl-tRNA synthetase tyrosyl-tRNA synthetase leucyl-tRNA synthetase aspartyl-tRNA synthetase alanyl-tRNA synthetase lysyl-tRNA synthetase arginyl-tRNA synthetase histidyl-tRNA synthetase phenylalanyl-tRNA synthetase alpha subunit Uncharacterized proteins putative uncharacterized protein putative uncharacterized protein Cell cycle cell division protein FtsZ septum site-determining protein MinD rod-shape-determining protein MreB (actin-like ATPase) Chaperones/Proteases ATP-dependent Clp protease ATP-binding subunit ClpX chaperone protein HtpG chaperone protein DnaK 60 kDa chaperonin trigger factor Metabolism aconitate hydratase 2 carbamoyl-phosphate synthase large chain ribonucleoside-diphosphate reductase ribose-phosphate pyrophosphokinase polyribonucleotide nucleotidyl transferase phosphoglycerate kinase phosphoenolpyruvate synthase

B7I353 D0CC75 D0CD22 D0CB16 D0CC02 B7GYM8 D0CAX2 D0CAX1 D0CCK6

ACIB5 ACIBA ACIBA ACIBA ACIBA ACIBA ACIBA ACIBA ACIBA

69/45/52 (57.07−62.12) 31/17/28 (49.14−68.73) 19/14/14 (46.57−58.51) 15/40/15 (15.60−33.00) 4/3/5 (15.34−30.21) 47/45/53 (57.87−60.11) 6/3/3 (7.69−14.57) 4/4/16 (6.67−20.47) 14/17/21 (19.52−39.42)

31/25/22 (37.37−56.31) 18/5/12 (23.37−48.11) 14/11/10 (47.76−52.84) 13/1/6 (1.43−14.75) 0/0/0 33/11/17 (21.77−48.60) 2/2/3 (5.06−17.40) 0/0/0 (2.02−11.60) 4/2/3 (6.42−12.06)

D0C7Q6 D0CCZ7 D6JS55 D6JTX8 D6JXA1 D6JXB0 F0QMZ5 D6JXA5 D0CCR5 D6JXA8 D0CDQ7 D0CDH4 D0S7T8 D0CAC9 D6JY12 F5JV30 D0C0J8 D0CEL9 F5JNX6 F5JNS9

ACIBA ACIBA ACIG3 ACIG3 ACIG3 ACIG3 ACIBD ACIG3 ACIBA ACIG3 ACIBA ACIBA ACICA ACIBA ACIG3 ACIBA 9GAMM ACIBA ACIBA ACIBA

39/46/45 (50.81−52.06) 15/11/16 (57.94−65.42) 18/9/4 (20.51−67.31) 12/10/14 (31.76−69.59) 4/2/2 (54.10−55.57) 9/14/4 (37.67−58.90) 6/2/4 (35.14−83.78) 5/3/6 (27.48−29.01) 5/6/4 (62.10−65.65) 13/8/3 (32.12−60.61) 5/3/5 (34.12−54.12) 6/9/10 (12.96−22.94) 3/5/7 (9.90−24.87) 7/8/4 (6.75−11.10) 5/6/5 (11.65−13.68) 6/9/6 (12.00−16.53) 10/11/13 (25.50−31.63) 13/14/16 (27.35−29.36) 2/3/3 (6.05−9.53) 3/3/2 (3.99−15.03)

18/17/21 (14.72−34.83) 8/9/9 (40.65−52.80) 4/3/3 (20.51−30.13) 9/8/8 (31.76−44.59) 0/0/0 3/3/6 (24.66−41.59) 0/0/0 0/0/0 0/0/0 2/2/3 (21.21−26.70) 1/2/2 (17.65−34.12) 0/0/0 3/2/2 (7.56−12.70) 3/2/2 (5.10−5.26) 3/2/2 (8.75−12.00) 0/0/0 4/3/3 (9.23−10.96) 4/2/4 (9.06) 0/0/0 0/0/0

D0C837 D0C837

ACIBA ACIBA

7/4/3 (11.50−31.94) 7/4/3 (11.50−31.94)

2/4/2(11.50−18.53) 2/4/2 (11.50−18.53)

D0CFL0 B0VTF1 F5JSE1

ACIBA ACIBS ACIBA

13/14/13 (38.11−43.48) 6/3/9 (20.74−37.89) 3/2/3 (9.25−13.01)

7/8/5 (20.97−23.02) 0/0/0 0/0/0

D0CAH0 F5I1R2 D0CFX9 D0CBD9 D0CAH2

ACIBA ACIBA ACIBA ACIBA ACIBA

4/5/5 (12.30−14.58) 7/10/15 (20.34−35.21) 24/37/31 (37.93−43.50) 43/36/49 (50.04−62.32) 25/23/26 (52.93−54.05)

0/0/0 0/0/0 18/20/18 (27.40−29.26 28/29/26 (48.99−50.92) 10/17/18 (33.33−41.67)

D0BWT5 D0CBB7 D0C9D5 D6JS83 D0CAU8 D0C7T5 B7IBB4

ACIBA ACIBA ACIBA ACIG3 ACIBA ACIBA ACIBA

24/28/37 (31.40−38.23) 22/20/15 (21.65−18.59) 8/6/4 (5.19−8.79) 13/4/7 (18.99−31.65) 10/14/16 (16.64−26.40) 7/3/6 (8.35−16.46) 23/24/24 (32.32−38.89)

16/7/8 (13.77−22.07) 5/2/1 (2.97−7.40) 0/0/0 0/0/0 9/7/9 (13.34−15.35) 3/2/3 (8.35−13.42) 18/11/12 (19.45−27.78)

F



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Table 1. continued

protein name



Downregulated proteins detected by the label-free quantitation technique succinyl-CoA ligase [ADP-forming] subunit β D0CDT5 ACIBA citrate synthase D0CDS6 ACIBA 1-pyrroline dehydrogenase D0CE42 ACIBA malic enzyme D0CC60 ACIBA Δ1-pyrroline-5-carboxylate dehydrogenase B2HYZ9 ACIBC argininosuccinate synthase B7I9G8 ACIB5 phosphoribosylformylglycinamidine cyclo-ligase D0CBK1 ACIBA 3-oxoacyl-[acyl-carrier-protein] synthase 2 D0C9P4 ACIBA ATP synthase subunit b D6JY66 ACIG3 succinate dehydrogenase, flavoprotein subunit D0CDS9 ACIBA aspartokinase E8PA67 ACIB1 CTP synthase D0C6Q3 ACIBA cytidylate kinase F5JLQ6 ACIBA pyrroloquinoline quinone biosynthesis protein PqqC F5JU55 ACIBA putative lytic murein transglycosylase, soluble (Slt) B0VQX3 ACIBS succinylornithine transaminase D0BYB3 9GAMM bifunctional protein GlmU D0CFE9 ACIBA aspartate ammonia-lyase (fumarate lyase; AspA) F5JM53 ACIBA TonB-dependent copper receptor (OprC) D0C582 9GAMM collagenase (peptidase, U32 family) F5JQ69 ACIBA NADH-quinone oxidoreductase subunit B (NuoB) F0QDV3 ACIBD NADH dehydrogenase subunit G NuoG F0QDV7 ACIBD uridylate kinase D6JTC2 ACIG3 SPFH domain-containing protein (band 7 protein; membrane F5JSG5 ACIBA protease subunit stomatin/prohibitin-like protein) phospho-2-dehydro-3-deoxyheptonate aldolase F5JLY2 ACIBA glutamate synthase subunit α (GltB) F5JSH9 ACIBA adenosylhomocysteinase B7I3L1 ACIB5 oligopeptidase A B7GW38 ACIB3 alanine racemase (sugar phosphate isomerase) D0CEQ8 ACIBA carbamoyl phosphate synthase small subunit F5JKV6 ACIBA γ-glutamyl phosphate reductase (proA) E8PBC6 ACIB1 fructose-bisphosphate aldolase D0S3G4 ACICA ubiquinol oxidase, subunit II D0C5Z5 ACIBA prephenate dehydratase F5JQ49 ACIBA aspartyl/glutamyl-tRNA amidotransferase subunit B F5JSD8 ACIBA transketolase B2HYZ1 ACIBC heat shock protein (fragment) D0C907 ACIBA threonine ammonia-lyase, biosynthetic D0C6N4 ACIBA rubredoxin−NAD(+) reductase F5IHI0 ACIBA dihydroxy-acid dehydratase F5JTU3 ACIBA inositol-1-monophosphatase D0S463 ACICA acyl-CoA dehydrogenase B7I8L8 ACIB5 glyceraldehyde-3-phosphate dehydrogenase D0S1I0 ACICA carbonic anhydrase F5JRE8 ACIBA 6,7-dimethyl-8-ribityllumazine synthase D0CFF3 ACIBA threonine synthase D0CB59 ACIBA enoyl-CoA hydratase/carnithine racemase D0S3I7 ACICA branched-chain amino acid aminotransferase D0CD87 ACIBA inosine-5′-monophosphate dehydrogenase D0CF46 ACIBA nucleotide triphosphate hydrolase domain-containing protein D0CCZ3 ACIBA ATP synthase F1, beta subunit D0CEK4 ACIBA bifunctional purine biosynthesis protein PurH D6JTZ1 ACIG3 phosphoribosylformylglycinamidine synthase PurL D0CBM2 ACIBA succinyl-CoA:coenzyme A transferase D0CCK3 ACIBA 2,3-bisphosphoglycerate-independent phosphoglycerate D0CB65 ACIBA mutase G

control

desiccation



in desiccation-stressed samples 31/21/16 (39.69−47.94) 16/11/16 (25.71−33.02) 4/8/7 (14.08−28.15) 12/16/18 (20.11−57.57) 13/17/7 (8.74−16.52) 12/6/12 (17.23−33.56) 7/4/3 (20.22−24.16) 6/8/10 (17.36−30.81) 8/3/6 (35.22−51.92) 14/13/15 (28.64−30.93) 2/2/2 (3.05−7.98) 7/5/6 (12.11−17.98) 2/2/2 (10.53) 2/2/2 (13.10−13.10) 2/2/2 (2.31−4.79) 4/3/3 (10.64) 4/2/3 (5.51−9.03) 2/2/2 (5.31) 3/2/2 (5.22−7.53) 2/2/2 (6.57) 2/2/3 (13.78) 12/7/10 (12.30−17.90) 8/5/6 (26.50−24.38) 3/2/2 (4.58−14.44) 3/2/2 (8.67−12.47) 2/4/2 (1.88−4.02) 5/6/2 (5.87−20.00) 5/3/8 (7.02−15.64) 5/2/2 (6.81−17.71) 4/2/3 (5.54−15.57) 2/2/4 (6.89−15.44) 8/4/6 (12.46−30.14) 5/3/2 (4.29−18.29) 4/3/3 (7.05−14.36) 5/3/2 (7.36−13.91) 3/6/2 (2.58−11.93) 8/6/7 (20.66−27.00) 4/4/3 (3.88−8.54) 3/2/6 (3.31−18.83) 2/3/6 (4.71−14.12) 5/3/4 (14.49−25.72) 4/7/4 (6.91−12.31) 13/15/11 (28.04−30.34) 6/4/2 (15.20−29.25) 6/3/3 (22.44−30.77) 12/9/7 (24.01−33.25) 6/2/2 (6.43−15.78) 10/5/9 (20.51−33.65) 8/7/11 (21.72−31.97) 5/4/4 (10.29−14.95) 26/19/16 (46.55−53.02) 6/7/8 (15.84−21.18) 15/14/12 (13.70−13.94) 4/5/5 (9.72−11.51) 7/3/6 (6.60−15.92)

15/11/14 (31.96-31.96) 11/8/9 (22.88−26.18) 5/3/3 (10.50−13.66) 7/8/5 (15.23−23.15) 0/0/0 2/2/3 (6.26−17.23) 0/0/0 2/2/4 (7.33−11.23) 13/8/11 (19.97−31.75) 0/0/0 5/3/4 (7.71−13.03) 0/0/0 0/0/0 0/0/0 2/2/2 (7.67) 2/2/2 (5.51) 0/0/0 0/0/0 0/0/0 0/0/0 2/2/2 (3.80) 5/2/4 (12.40−24.30) 0/0/0 0/0/0 0/0/0 2/1/3 (3.04−17.60) 3/2/2 (1.30−5.70) 0/0/0 0/0/0 0/0/0 4/2/2 (13.63−27.80) 0/0/0 0/0/0 0/0/0 0/0/0 2/4/2 (6.89−15.23) 0/0/0 0/0/0 0/0/0 0/0/0 2/2/2 (3.54−9.40) 10/7/10 (18.76−23.30) 0/0/0 0/0/0 6/3/5 (12.14−23.48) 0/0/0 3/2/5 (11.54−20.51) 5/3/5 (8.40−17.62) 0/0/0 14/7/15 (24.14−45.47) 3/2/2 (7.44−11.30) 5/2/2 (6.50−7.10) 0/0/0 2/1/1 (2.33−4.27)



Article

Table 1. continued

protein name



Downregulated proteins detected by the label-free quantitation technique cysteine synthase F5IN91 ACIBA tail-specific protease D0CAF5 ACIBA homoserine dehydrogenase D6JV16 ACIG3 aspartate-semialdehyde dehydrogenase D0S5A0 ACICA ketol-acid reductoisomerase D6JVT7 ACIG3 aspartyl/glutamyl-tRNA amidotransferase subunit A F5JSD9 ACIBA adenylsuccinate lyase D0CBY3 ACIBA aminopeptidase N B0VM88 ACIBS peptidase D0CBV3 ACIBA short-chain dehydrogenase/reductase D0C6A8 ACIBA (acyl carrier protein) S-malonyltransferase F5JLG9 ACIBA putative uncharacterized protein (phosphoglycerate D6JX16 ACIG3 dehydrogenase) 2-oxoglutarate dehydrogenase, E2 component, D0CDT3 ACIBA dihydrolipoamide succinyltransferase phosphoribosylaminoimidazole-succinocarboxamide synthase D6JW93 ACIG3 glutamine-fructose-6-phosphate transaminase D0CFE8 ACIBA isocitrate dehydrogenase [NADP] D0CBU4 ACIBA glutamine synthetase D0CC50 ACIBA dihydrolipoyl dehydrogenase D0CDT4 ACIBA 3-ketoacyl-CoA thiolase F5JPX6 ACIBA ATP synthase subunit α D0CEK6 ACIBA fatty acid oxidation complex subunit α D0CAZ9 ACIBA nucleoside-diphosphate sugar epimerase D0C3F0 9GAMM ABC transporter ATPase D0CCL0 ACIBA a

control

desiccation



in desiccation-stressed samples 6/3/5 (10.54−34.64) 6/7/14 (11.69−23.52) 6/7/2 (19.40−24.26) 7/5/3 (11.56−25.54) 21/7/8 (24.56−61.54) 5/5/6 (13.01−14.84) 6/8/4 (12.55−27.06) 9/9/13 (11.52−20.38) 4/4/9 (5.87−15.11) 8/5/6 (12.31−24.19) 7/5/7 (20.54−19.82) 17/11/13 (21.71−40.73)

0/0/0 7/1/3 0/0/0 0/0/0 4/1/4 0/0/0 0/0/0 5/4/4 0/0/0 0/0/0 0/0/0 7/4/9

(3.03−18.20)

(5.62−18.64)

(7.03)

(11.71−21.46)

14/14/20 (22.64−30.64)

10/7/10 (14.32−20.35)

8/7/2 (6.70−31.38) 5/10/4 (11.60−23.69) 12/13/16 (19.06−26.17) 17/14/19 (20.08−25.51) 26/18/16 (37.32−45.74) 7/10/15 (20.34−35.21) 30/24/28 (41.23−46.44) 18/15/23 (28.03−41.42) 23/13/18 (13.46−41.87) 9/9/9 (19.71−22.60)

0/0/0 0/0/0 11/4/5 (6.71−17.05) 11/5/6 (15.29−20.08) 13/7/11 (21.59−38.57) 0/0/0 18/6/14 (13.87−35.26) 9/1/3 (2.65−19.11) 0/0/0 5/2/5 (5.42−10.49)

Proteins detected by both proteomics approaches used in this study are shown in bold type.

Blast2Go Analysis

component 1 = 0.410XC1 + 0.361XC2 + 0.400XC3

The results obtained by both proteomics approaches were combined and analyzed with the aid of Blast2GO software.13 The results for differentially expressed proteins are shown in Figure 3. In the “Biological Process” section, the proteins from the control samples included translation, small molecule catabolic process, and cellular amino acid biosynthesis processing, among others, suggestive of active metabolism (Figure 3A1). In the desiccation-stressed samples, the overexpressed proteins were associated with processes such as transport, oxidation−reduction process, and response to oxidative stress/chemical stimulus (Figure 3B1). In the graph showing “Molecular Function” of the control samples, overrepresented proteins were included in the categories RNA binding and ATP binding, among others (Figure 3A2), and in the desiccation-stressed samples, the functions detected included DNA binding, transporter activity, and peroxidase activity (Figure 3B2). Considering “Cellular Component”, over-represented proteins appeared in the intracellular organelle part, ribosome, and protein complex in the control samples (Figure 3A3), whereas in the desiccation-stressed samples, the overexpressed proteins were included in the cytoplasm, membrane, and outer membrane-bound periplasmic space (Figure 3B3). Since clear differences were found relative to cellular localization, different bioinformatics tools were used to identify putative export signals in proteins overexpressed in the desiccated samples, including catalase HPII as a known secreted protein and the ribosome recycling factor as a negative control.

+ 0.434XD1 + 0.413XD2 + 0.427XD3 component 2 = 0.399XC1 + 0.463XC2 + 0.410XC3 − 0.344XD1 − 0.436XD2 − 0.387XD3

where C1, C2, and C3 denote the results for each of the control samples, and D1, D2, and D3 denote the samples after being subjected to desiccating conditions. Therefore, the first component represents a weighted average of the six experimental values and distinguishes proteins by their average overall expression over the six experiments. The second component distinguishes proteins according to the difference in the number of peptides in control and desiccation-stressed samples. A larger positive loading in the second component indicates that the protein is overexpressed in the control relative to the desiccation-stressed samples. On the contrary, negative values in the second component indicate that the protein reaches higher concentrations in desiccation-stressed samples. The score plot shows the projections of the data on the first (x axis) and second (y axis) principal components, in which each point represents a protein (Figure 2 in Supporting Information). Those proteins displaying most differences in control and desiccation-stressed samples are furthest from the horizontal axis. H



Article

Table 2. Proteins Quantified by iTRAQ (P < 0.05)a Uniprot accession number

protein name

Upregulated proteins in desiccation-stressed samples Transcriptional regulators integration host factor subunit β D0C7Q5 DNA-binding protein HU D0S3N3 Translation ribosome-recycling factor D0C6G8 Proteins related to antimicrobial resistance β-lactamase ampC Q7WZ37 carbapenem-associated resistance protein CarO C0L0K3 putative outer membrane protein Omp25 Q4A208 AdeI Q2FD95 alkyl sulfatase D0SNQ0 uncharacterized protein tol-pal system protein YbgF D0C6H8 ROS RNS detoxifacition catalase HPII (hydroperoxidase) B7I3G1 superoxide dismutase [Cu−Zn] D0CCT4 peroxiredoxin D0CDD0 UvrABC system protein B D0CBR9 glutathione peroxidase B7I1W7 glutaredoxin-4 D0C6L1 flavohemoprotein D0CCZ2 Chaperones ClpB. ATP-dependent protease, Hsp 100, part of multichaperone system with DnaK, D0CEH1 DnaJ, and GrpE GrpE B7IBK6 peptidyl-prolyl cis−trans isomerase (SurA) D0C7T2 host factor Hfq B7IAP5 chaperone protein Skp D0C6H4 Toxin putative uncharacterized protein (CspD) D6JU64 Uncharacterized proteins putative uncharacterized protein D0CFS8 putative uncharacterized protein D0C5R3 uncharacterized conserved protein B2HUF9 putative uncharacterized protein D0C9P5 putative uncharacterized protein D0C640 putative uncharacterized protein D0C780 putative uncharacterized protein D0CDW4 Adherence and motility type VI secretion system OmpA/MotB D0C9R5 NlpE D0C8Y7 CsuA/B D0BX46 Metabolism ATP synthase subunit b D6JY66 succinate dehydrogenase, flavoprotein subunit D0CDS9 Zn-dependent hydrolase, including glyoxylase B2HXB4 alcohol dehydrogenase D6JTQ2 aspartate/tyrosine/aromatic aminotransferase B2HUD5 aconitate hydratase 1 D0BVW6 isochorismatase hydrolase B7I8E7 formaldehyde dehydrogenase, glutathione-independent D0C8R0 dehydrogenase with different specificities B2HY96 NAD-dependent aldehyde dehydrogenase B2HTS2 putative intracellular protease/amidase B2I223 GTPase Obg D0CBS5 isocitrate lyase D0C8Y6 Secreted proteins putative 17 kDa surface antigen B2HY91 LysM domain-containing protein D0C9K9

I

peptides (95%) (% coverage)

117:114 ratio

117:114 P value

11 (89.0) 15 (86.7)

1.34 1.21

0.0339 0.0270

17 (76.6)

1.24

0.0007

64 (63.9) 14 (45.6) 7 (41.2) 3 (27.4) 7 (25.2) 4 (18)

29.38 5.81 1.49 5.20 1.74 1.59

0.0072 0.0263 0.0015 0.0490 0.0055 0.0500

51 (56.7) 4 (42.4) 8 (45.1) 5 (25.0) 9 (68.0) 9 (52.4) 9 (39.8)

2.16 2.14 1.29 1.32 1.45 1.54 1.26

0.0000 0.0046 0.0418 0.0136 0.0153 0.0042 0.0089

38 (61.5)

1.22

0.0069

11 10 17 15

(51.1) (43.5) (47.1) (79.6)

1.23 1.27 2.17 1.34

0.0248 0.0024 0.0008 0.0023

11(70.0))

1.89

0.0043

33 (67.5) 70 (100.0) 6 (72.5) 6 (30.6) 5 (34.5) 3 (54.2) 5 (37.1)

3.50 2.56 2.60 1.41 2.28 4.34 1.22

0.0000 0.0003 0.0014 0.0097 0.0112 0.0399 0.0413

26 (76.0) 8 (59.1) 8 (37.2)

1.31 1.73 1.69

0.0047 0.0391 0.0169

9 (59.0) 28 (62.7) 4 (28.1) 7 (25.9) 8 (63.2) 23 (37.5) 6 (34.8) 4 (23.5) 19 (65.4) 12 (35.8) 6 (42.6) 9 (47.3) 12 (34.6)

1.33 1.31 1.42 2.77 2.01 1.39 1.83 1.45 2.24 2.78 3.15 1.29 1.41

0.0080 0.0176 0.0281 0.0282 0.0416 0.0021 0.0445 0.0475 0.0000 0.0005 0.0104 0.0203 0.0247

44 (75.2) 6 (44.0)

2.48 1.56

0.0006 0.0055



Article

Table 2. continued Uniprot accession number

protein name LemA family protein YceI family protein RNA processing ribonuclease E Proteins related to quorum quenching dihydrocoumarin hydrolase dienelactone hydrolase

Upregulated proteins in desiccation-stressed samples D0CCP5 D0C7 V7 B7I4C6 Q83WC8 D0CFB4 Downregulated proteins in desiccation-stressed samples

Transcription elongation factor Tu translation elongation factor Ts DNA-directed RNA polymerase subunit alpha Translation ribosomal protein S1 30S ribosomal protein S7 50S ribosomal protein L14 50S ribosomal protein L3 30S ribosomal protein S6 Cell cycle cell division protein FtsZ septum site-determining protein MinD Chaperones/Proteases ATP-dependent Clp protease ATP-binding subunit ClpX ATP-dependent Clp protease ATP-binding subunit ClpA Metabolism aconitate hydratase 2 succinyl-CoA ligase [ADP-forming] subunit β citrate synthase 1-pyrroline dehydrogenase malic enzyme Δ1-pyrroline-5-carboxylate dehydrogenase argininosuccinate synthase phosphoribosylformylglycinamidine cyclo-ligase 3-oxoacyl-[acyl carrier protein] synthase 2 ErfK/YbiS/YcfS/YnhG family malate dehydrogenase, NAD-dependent tyrosine aminotransferase tyrosine repressible acetyl/propionyl-CoA carboxylase, α subunit fumarate hydratase class I 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase 4-hydroxyphenylpyruvate dioxygenase acetyl-coenzyme A carboxylase carboxyl transferase subunit α adenylsuccinate synthetase putative acetyl-CoA hydrolase/transferase glucose-6-phosphate isomerase Uncharacterized proteins putative uncharacterized protein putative uncharacterized protein putative uncharacterized protein Recombination protein RecA a

peptides (95%) (% coverage)

117:114 ratio

117:114 P value

7 (58.7) 7 (44.4)

1.47 1.90

0.0060 0.0007

7 (32.9)

1.77

0.0126

13 (42.) 8 (14.3)

3.92 2.00

0.0029 0.0218

0,48 0.79 0.78

0.0000 0.0000 0.0223

B7I353 D0CC75 D0CD22

104 (81.1) 42 (92.4) 59 (73.1)

D0BYX9 D6JS55 D6JXA1 D0CCZ7 D0C5Z0

53 19 10 24 18

(67.5) (81.4) (85.3) (73.8) (80.3)

0.55 0.67 0.77 0.65 0.67

0.0000 0.0154 0.0490 0.0021 0.0231

D0S4 V4 D0C9R1

16 (56.3) 14 (61.2)

0.69 0.71

0.0302 0.0498

D0CAH0 D0C6P2

5 (23.3) 7 (28.0)

0.38 0.66

0.0164 0.0208

D0BWT5 D0CDT5 D0CDS6 D0CE42 D0S780 B2HYZ9 B7I9G8 D0CBK1 D0C9P4 D0CC33 D0CD53 D0CET6 B2HXI4 D0CAG7 D0CBN5 D0CFC8 B2HTI7 D0CEG3 B0VPC7 B7H2H4

44 (57.1) 44 (67.5) 29 (62.7) 8 (31.7) 5 (22.3) 16 (36.8) 14 (52.8) 10 (45.8) 13 (38.9) 11 (24.2) 57 (81.1) 6 (31.9) 11 (39.7) 14 (38.0) 19 (58.2) 5 (25.9) 13 (49.8) 10 (39.9) 6 (28.8) 4 (15.9)

0.64 0.70 0.76 0.62 0.70 0.66 0.79 0.74 0.62 0.59 0.78 0.45 0.56 0.51 0.77 0.37 0.69 0.72 0.41 0.75

0.0000 0.0004 0.0027 0.0051 0.0055 0.0236 0.0291 0.3830 0.0500 0.0001 0.0006 0.0007 0.0000 0.0021 0.0034 0.0079 0.0186 0.0317 0.0500 0.0500

D0CFJ8 D0CBY9 D6JWH2

8 (54.5) 3 (36.4) 18 (67.2)

0.71 0.54 0.75

0.0060 0.0203 0.0312

D0C6I0

6 (42.7)

0.69

0.0200

Proteins detected by both proteomics approaches used in this study are shown in bold type.

tion, which affect a large number of proteins such as outer membrane proteins, efflux pumps, tol-pal system, members of the Sec secretion system, and proteins like OmpA/MotB that are part of the type VI secretion system. We also found a considerable number of secreted proteins, as predicted by

As shown in Table 3, in most of the proteins, a putative export signal was identified suggesting membrane, periplasmic, or extracellular targeting. The modifications that A. baumannii undergoes as a result of stress by desiccation include changes in membrane composiJ



Article

Figure 3. Gene ontology categories after Blast2Go analysis of all differentially expressed proteins detected by label-free and iTRAQ quantitation analyses. A represents overexpressed proteins in the control sample, and B represents overexpressed proteins in the dessication conditions. (1) Biological Process; (2) Molecular Function; and (3) Cellular Component.

conidiation-specific protein 10 family protein, or the LysM domain-containing protein. The RNA expression and proteomics analyses revealed similar patterns for porins such as Omp25, and CarO and the toxins hemolysin and septicolysin. However, RNA expression of DcaP-like, the efflux pump adeJ, as well as catHPII was not consistent with the protein results, probably because of post-transcriptional regulation.

bioinformatics applications and literature search, although most of them are of unknown function. We conclude that membrane permeability is changed by nutrient shortage and limited water availability. Besides, we detected other ongoing processes including the packing of the chromosome mediated by HU (a histone-like protein from Escherichia coli) and IHF (integration host factor); a general decrease in transcription and translation rates; alterations in cell division as determined by the reduction of FtsZ, MreB, or MinD proteins or overproduction of reactive oxygen species, which can be considered as global markers of stress. Other processes include an increase in chaperones such as Hfq (which stabilizes certain mRNAs), ClpB (which stabilizes proteins and abolishes protein aggregation in association with GrpE), and Skp (described as a stabilizer of outer membrane proteins). Another characteristic of the system is the increase in proteins involved in quorum quenching, such as dienelactone hydrolase and Zn-dependent hydrolase (including glyoxylase). Moreover, there was also an increase in proteins associated with antimicrobial resistance, such as AmpC and the efflux pump AdeIJK, the tol/pal system, and SdsA, which is able to increase resistance to sodium dodecyl sulfate and other detergents.

Biofilm and Electron Microscopy Analysis

Since the inclusion of bacterial cells within a biofilm matrix has long been considered a protective factor against environmental insults, control and desiccation samples were stained with crystal violet. The control sample did not show significant differences in absorbance related to the blank control, while the desiccation sample showed absorbance values consistent with the deposition of a biofilm matrix (Table 4). Scanning electron microscopy confirmed the presence of a thick layer of biofilm matrix surrounding the dormant cells, in opposition to the controls (Figure 5A). Control samples and those subjected to desiccation for 30 days were analyzed by scanning electron microscopy, which revealed clear morphological changes in the cells: the control cells were typically rod-shaped cells, and the desiccationstressed cells were spherical (Figure 5B,C). Transmission electron microscopy of ultrathin sections showed the appearance of electrondense aggregates in the inside of dormant cells (Figure 5D), in opposition to the more homogeneous aspect of the cytoplasm of the control cells.

RT-PCR

RT-PCR analyses were carried out on 16 genes, and it was found that the expression of most, but not all, paralleled the expression of the corresponding proteins seen in proteomic analysis (Figure 4). Expression of the β-lactamases oxa51 and ampC was higher in desiccation-stressed samples than in the control samples, as previously found in our proteomics studies. A similar situation occurred with proteins with a predicted signal peptide, such as TolA, putative 17 KDa surface antigen,

■

DISCUSSION Bacteria can survive drastic changes in intracellular osmolarity by responding both passively and actively to changes in the K



Article

Table 3. Analysis of the Existence of a Signal Peptide in Proteins Upregulated or Downregulated in Samples Subjected to Desiccationa name Catalase HPII ribosome-recycling factor putative uncharacterized protein putative uncharacterized protein septicolisyn glutathione peroxidase peroxiredoxin putative uncharacterized protein (OB-fold) surface antigen superoxide dismutase putative uncharacterized protein LysM domain-containing protein rhodanase domain protein Ycel family protein LemA family protein uncharacterized conserved protein putative uncharacterized protein putative uncharacterized protein putative uncharacterized protein putative uncharacterized protein type VI secretion system OmpA/ MotB alcohol dehydrogenase metallo-β-lactamase superfamily protein dihydrocoumarin hydrolase dienelactone hydrolase putative uncharacterized protein putative uncharacterized protein putative uncharacterized protein

accession number (Uniprot)

export signal

D0C8B2 D0C6G8 D0C5R3 D0CFS8 B7I863 B7I1W7 D0CDD0 D0C5R8

+1 − +1 +1, 2 − + + +1

D0C8B6 D6JUJ4 D0CBN2 D0C9K9 B7I988 D0C7 V7 D0CCP5 B2HUF9 D0C9P5 D0C640 D0C780 D0CDW4 D0C9R5

+1 +1 +2 + +1 +2 +2 +1 + − + − +1, 2

D6JTQ2 B2HXB4

− +1, 2

Q83WC8 D0CFB4 D0CFJ8 D0CBY9 D6JWH2

+1 + +1 +1, 2 −

Table 4. Crystal Violet Staining Analysis of Biofilm Formation conditions

abs 570 nm mean (SD)

control AbH12O-A2 desiccation AbH12O-A2 blank

0.09 (0.03) 0.84 (0.05) 0.11 (0.02)

a

Proteins with (+) or without (−) export signal, as predicted by a combination of software. Protein detected in the secretome:39 1, in the soluble fraction (FSEP); 2, in the outer membrane vesicle (OMV) fraction. Proteins upregulated in desiccating conditions are shown in bold case.

Figure 5. Scanning electron microscopy images at different magnifications showing the accumulation of biofilm in control cells (A1) or after 30 days in desiccation conditions (A2), and the changes in morphology in control (B1,C1) and desiccated samples (B2,C2) of A. baumannii strain AbH12O-A2 cells. Transmission electron microscopy (60 000×) images reveal homogeneous cytoplasm in control cells (D1) and the accumulation of electrodense material in desiccated bacterial cells (D2).

environment. In particular, members of the genus Acinetobacter are well-known for their metabolic versatility that allows them to adapt to different ecological niches.17 From our results, we conclude that cells maintained in prolonged desiccation conditions are cells in a dormant state. In Figure 1, it is shown that, from the seventh day of desiccation, a population of around 0.5% of the original population remained alive for at least 3 weeks more and is able to resume growth in more favorable conditions. Interestingly, A. baumannii ATCC 17978 and AbH12O-A2 strains had a similar growth curve in desiccation conditions, possibly due to the fact that both are poor biofilm-forming strains. In this sense, strains of A. baumannii exhibit different behavior under desiccation conditions depending on if they are poor biofilm formers or if they form an abundant extracellular matrix. 18 Two proteomics approaches were used to study how A. baumannii

Figure 4. RT-PCR analysis of different genes coding for proteins differentially expressed between control samples and desiccationstressed (dormant) samples. All expression results were normalized against gyrB by the 2−ΔΔCt method. For all genes, relative mRNA expression is presented as a fold-change value over the control sample. catHPII, catalase HPII; dhch, dihydrocoumarin hydrolase; surgAg, putative 17 kDa surface antigen; cnsp, conidiation-specific protein 10 family protein.

L



Article

Figure 6. Working model showing the molecular mechanisms elicited after desiccation of the A. baumannii AbH12O-A2 strain that may underlie the development and maintenance of a dormant phenotype in persistent cells. These mechanisms include changes in the composition of membrane proteins, as well as changes in DNA organization and in transcriptional regulation, resulting in the overexpression of protein chaperones and antimicrobial resistance-related proteins among others. The dashed line encloses known effectors of the Cpx pathway, suggesting its involvement in the development of dormancy. Maroon, upregulated proteins in desiccation conditions, as detected by both label-free quantitation and iTRAQ; dark blue, upregulated proteins detected by label-free quantitation; dark green, upregulated proteins detected by iTRAQ; red, downregulated proteins in desiccation conditions, as detected by both label-free quantitation and iTRAQ; red, downregulated proteins detected by label-free quantitation; bright blue, downregulated proteins detected by iTRAQ; (→) presence of an export signal.

cells have been shown to be abundant in bacterial biofilms.23 This, together with the growth curves seen along the desiccation treatment, leads us to think that a dormant phenotype had been selected in our system. Regarding chromosome organization, we observed a decrease in expression of the elongation factor TU, elongation factor P, RpoA, and RpoC. Conversely, we found overexpression of two members of the DNABII family of DNA-binding proteins in desiccation-stressed samples, IHF and HU, which are involved in transcription regulation and are essential for maintaining DNA supercoiling and compaction.24 It is conceivable that increased packing of the chromosome is an acquired strategy to protect the hereditary material in adverse conditions. The HU protein also coordinates the expression of genes involved in central metabolism and virulence and contributes to the success of Salmonella enterica as a pathogen.25 Consistent with this, transmission electron microscopy images show the presence of an electrodense material in the inside of desiccation-stressed A. baumannii cells, as previously reported.18,26 At the translational level, we found increased expression of the ribosomal recycling factor (RRF) in the desiccation-stressed samples. Disassembly of the ribosome at the end of translation is facilitated by the RRF. In addition, a role in error reduction during peptide elongation has been proposed.27 We also found a general decrease in expression of proteins associated with the ribosome in the desiccation-stressed samples, suggestive of a

is able to survive extreme conditions. First, we used a label-free quantitation methodology, which produced a semiquantitative measurement through the number of peptides detected. Although ESI-MS/MS has been the preferred technique for several years, recent advances in the reproducibility of the chromatographic systems and batch-data acquisition allow the use of LC-MALDI-TOF/TOF systems in label-free quantitation.10,19,20 The second approach was based on an iTRAQ labeling of the samples and direct quantification of each protein. It has been previously shown that the combination of different proteomic approaches results in identification of more proteins and higher coverage.21 This work shows important changes between the proteomes of planktonic cells or cells undergoing prolonged desiccation. Gene ontology categories were assigned to the proteins found to be under- or overexpressed in the desiccation samples. On the basis of the above results, as well as after extensive revision of published information on the known function of modulated proteins, we propose a working model (Figure 6) that includes proteins involved in the molecular mechanisms triggering the persistence phenotype, with tight regulation at different levels. A general decrease in expression of proteins involved in transcription and translation was found, as expected in a spore-like state. Interestingly, most of the proteins overexpressed in desiccation-stressed conditions had been previously found in dormant persistent cells.22 Also, dormant M



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reduced rate of translation and protein synthesis and an increased control over the existing translation. At a second level of post-translational regulation, the chaperones GrpE, ClpB, Hfq, and Skp were overexpressed in desiccation-stressed samples. ClpB cooperates with the DnaK.JGrpE set in the recovery of several types of heat-inactivated proteins,28 which may be involved in the prevention of protein aggregation.29 The involvement of a ClpB chaperone in virulence and stress resistance has previously been reported in the pathogen Leptospira interrogans.30 A ClpB null mutant also showed an impaired ability to replicate under permissive growth conditions.31 Hfq has been shown to interact with small regulatory RNAs and act as a post-transcriptional regulator of mRNA stability and translation. Hfq mutants often exhibit pleiotropic phenotypes involved in virulence, quorum sensing, growth rate, and stress tolerance.32 Finally, Skp has been reported to be involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.33 Inactivation of Skp has been reported to result in a reduction of outer membrane proteins.34 The concurrent expression of this set of chaperones might be seen as a molecular mechanism to prevent aggregation and ensure correct protein folding and targeting in conditions of increased osmotic pressure. Regarding the antioxidant system, we observed induction of proteins, such as glutathione peroxidase, catalase H, or superoxide dismutase in the desiccation-stressed samples. Proteins like alkylhydroperoxide reductase and catalase are required for environmental persistence and nasal colonization of Staphylococcus aureus.35 Thus, oxidative stress resistance is an important factor in the ability of bacteria to persist in the hospital environment and thus contributes to the spread of human disease. Some of the proteins overexpressed in desiccation-stressed samples are associated with antimicrobial resistance. For example, AmpC and Oxa51 are involved in antibiotic resistance. There was also an increase in the production of some outer membrane proteins (Omp25, DcaP-like and CarO), which indicated a change in membrane composition and permeability owing to the extreme environmental conditions of the experiment. In this sense, it is also noticeable the thickening of the cell wall of dormant cells, as observed in transmission electron microscopy images by us and others.18,26 Besides, we observed activation of the efflux pump AdeIJK, which has been reported to contribute to the resistance to numerous antibiotics, including β-lactams, chloranphenicol, and tetracycline.36 In addition to the above, we found overexpression of the protein alkyl sulfatase, which has been reported to be involved in the degradation of anionic surfactants.37 The protein TolA is also overexpressed in desiccating conditions. Previous studies have shown its involvement in membrane impermeability protecting against the entry of antibiotics in Pseudomonas aeruginosa. This protein alters the lipopolysaccharide structure, resulting in a reduced affinity of aminoglucoside antibiotics for the outer membrane.38 Our results were analyzed using the Blast2Go program, and we found an increase in proteins located in the periplasmic space and the membrane in the desiccation samples (Figure 3). We then performed extensive in silico analysis to identify secretion signals, shown in Table 3. We found that most of the analyzed proteins have secretion signals. Actually, some of them have been identified in a study of the secretome of this strain.39

Repression of proteins involved in cell division was also observed in the desiccation-stressed samples, for example, of FtsZ, a tubulin-like responsible for the formation of the septum during cell division. This may be related to overproduction of Hfq, which binds the processing site of FtsZ and inhibits its translation, resulting in inhibited cell division because of decreased expression of FtsZ.40 Moreover, FstZ function is negatively regulated by the MinCD complex. The MinD protein, which is underexpressed in desiccating conditions, is located at cell poles and determines the position where the cell division septum must be synthesized. Polymerization of FtsZ can strangle the membrane and contribute to the constriction process. A mutation in this gene abolishes cell division.41,42 One interesting result, which may explain the change in the morphology of A. baumannii, was the underexpression in desiccating conditions of the rod-shape-determining protein MreB (actin-like ATPase). MreB is a major homologue of actin in terms of primary sequence, structure, and size.43 When this protein is depleted, rod-shaped Bacillus subtilis and E. coli cells become spherical.44,45 A decrease in expression of this protein may explain the spherical shape adopted by the A. baumannii cells subjected to desiccating conditions, as observed by scanning microscopy (Figure 5). Möker et al.46 showed that, although P. aeruginosa cultures contain a small number of “persister” cells, these can increase in number in response to quorum sensing related signaling molecules, such as autoinducers. Quorum sensing is the intracellular communication between cells of a population, and it involves cooperative behavior. Quorum quenching is the inhibition of quorum sensing by degradative enzymes that target signaling molecules. In the present study, we found that the enzyme dienelactone hydrolase (Dhlr) was overexpressed in the desiccation-stressed sample. Krysciak et al.47 showed that Dhlr inhibited biofilm formation and other quorum-sensingdependent processes in P. aeruginosa, Chromobacterium violaceum, and Agrobacterium tumafaciens. These authors also demonstrated that members of the metallo-β-lactamase superfamily function as acyl homoserine lactone (AHL) lactonases. In the present study, we found that a member of the metallo-βlactamase superfamily, Zn-dependent hydrolase, including glyoxylase, was overexpressed in the desiccation-stressed samples. This protein (320 amino acids with a conserved motif HLHPDH∼H) has been described in the literature as belonging to a domain of AHLases. AhlD shared the same HXHXDH∼H pattern, referred to as the zinc metallohydrolase criterion.48 Remarkably, several proteins related to the Cpx pathway have been identified in this study. The Cpx two-component signal transduction system controls a stress response and is activated by misfolded proteins in the periplasm. This pathway has been shown to mediate abiotic hydrophobic surface adhesion in E. coli.49,50 Although the ClpX protein itself was not detected, one of its main effectors, NlpE, was found to be overexpressed in the iTRAQ analysis of desiccated samples. Besides, a considerable number of well-characterized Cpx-responsive proteins (Skp, SurA, CarO, MinD, Omp25, surface antigen, Csu A/B, CsuC, MviM) were also identified. These findings raise the possibility that the Cpx pathway may be involved in the maintenance of a resistant dormant phenotype. One of the best studied genes in relation to persistance is HipA. This gene is part of the operon HipA/B, a bacterial toxin−antitoxin system, with HipA being the toxin. The increase in expression of HipA can induce dormancy.51 Some N



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scopiá Servizos de Apoio á Investigación, Universidade da Coruña) for her valuable assistance in the processing of SEM samples, and to Sonia Pertega (Unidad de Estadistica of the CHUAC) for her assistance with data analysis. Critical reading of the manuscript by Nelson C. Soares (University of Cape Town, Institute of Infection Disease and Molecular Medicine, IIDMM) and Diego Garciá (Health in Code) is greatly appreciated. M.T. was financially supported by the Miguel Servet Programme (C.H.U.A Coruna and ISCIII). This work was funded by the Spanish Network for the Research in Infectious Diseases-REIPI-(Instituto de Salud Carlos III, RD12/00552), FIS PI081613, PI10/00056, PS09/00687, PS07/90, PS07/51, PI12/00552, and 08CSA064916PR from Xunta de Galicia. This study was also funded by grants from the European Community, FP7, ID: 278232 (MagicBullet).

authors have shown involvement of HipA in the arrest of the cell cycle, and recent studies suggest that HipA is a kinase that inhibits translation by phosphorylating the EF-TU elongation factor required for growth arrest and multidrug tolerance.52 Both blocking of cell division and quantitative decrease of the elongation factor EF-Tu were observed in our desiccation study. Our results point to the involvement of HipA in the development of persistence. Only recently, a direct link was provided between the recalcitrance of chronic infections and persistence.53 Leung and Lévesque54 demonstrated that “persister” cells are formed after exposure of Streptococcus mutans, the main etiological agent of dental caries, to a wide range of environmental conditions, fluctuations in pH, and high levels of salt from tooth demineralization. Although significant work has been done regarding dormancy in environmental microbial samples, ours is, as far as we know, the first study demonstrating selection of persistent cells by environmental stress on inanimate surfaces in the context of nosocomial infection. We conclude that, in the first stage, within the first 5 days after plating a highly concentrated culture of A. baumannii AbH12O-A2, a biofilm was formed. However, when the population was maintained under prolonged stress, a small subpopulation comprising around 0.5% of the original population, which was tolerant to extreme desiccation, was selected. Upon nutrient exhaustion, the few viable surviving bacteria subsist on the corpses of their siblings and, when environmental conditions become more favorable, the surviving bacteria resume growth and restore the population. Considering that A. baumannii is not a spore-forming bacterium, we believe that the adoption of this “bust-and-boom” strategy could explain the ability of a few surviving bacterial cells to infect immunocompromised patients, explaining the recurrent outbreaks found in ICU settings. In this sense, previous work by Balaban et al.55 showed that the inherent heterogeneity of bacterial populations may be important regarding adaptation to fluctuating environments and in the persistence of bacterial infections. This mechanism could explain the maintenance of latent bacterial populations in hospital settings. We propose that formation of A. baumannii dormant cells may represent a general survival mechanism in conditions of desiccation and lack of nutrients, as found in ICUs, which may result in a chronic presence in ICUs with periodic outbreaks.

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S Supporting Information *

Additional tables and figures as described in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address §

Health in Code, Xubias s/n, 15006 La Coruña, Spain.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS ́ We are grateful to Manuel José Gómez Rodriguez (Centro de Astrobiologia,́ CSIC) for his invaluable assistance with bioinformatics, to Ada Castro Couceiro (Unidad de MicroO



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