Curr Microbiol (2014) 68:352–357 DOI 10.1007/s00284-013-0483-6
Study of Lysozyme Resistance in Rhodococcus equi Laurent He´bert • Pauline Bidaud • Didier Goux Abdellah Benachour • Claire Laugier • Sandrine Petry
•
Received: 28 June 2013 / Accepted: 17 September 2013 / Published online: 30 October 2013 Ó Springer Science+Business Media New York 2013
Abstract Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.
L. He´bert P. Bidaud C. Laugier S. Petry (&) Dozule´ Laboratory for Equine Diseases, Bacteriology and Parasitology Unit, ANSES, 14430 Goustranville, France e-mail: [email protected] L. He´bert e-mail: [email protected] D. Goux UNICAEN, CMAbio, Normandie Univ, F-14032 Caen, France A. Benachour UNICAEN, U2RM, Normandie Univ, F-14032 Caen, France
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Introduction Rhodococcus equi is a facultative intracellular multihost pathogen affecting mainly foals up to 6 months of age which can cause severe chronic suppurative bronchopneumonia with abscessation, as well as clinical signs of intestinal disease and occasionally septic arthritis or osteomyelitis [7]. In addition, R. equi is an opportunistic zoonotic pathogen commonly identified in immunocompromised people, particularly in patients with AIDS and organ transplant recipients [15, 19], and more rarely in immunocompetent patients [11]. In general, the pathogenicity of R. equi is conferred by the presence of an 80–90 kb virulence plasmid [24] that enables the survival and replication of the bacteria within a membrane-bound vacuole inside macrophages [10]. To survive and colonize a given host, a pathogen must successfully overcome the constitutive or innate defense system and the host’s phagocytic response. One of the most important and widespread components of this defense system is lysozyme. This compound is found in a wide variety of body fluids, such as respiratory and saliva secretions, as well as in cells of the innate immune system, including neutrophils, monocytes, macrophages, and epithelial cells [4, 5]. Lysozyme is an enzyme that cleaves peptidoglycan (PG) between the glycosidic beta-1,4-linked residues of N-acetylmuramic acid and N-acetylglucosamine resulting in the degradation of the PG, and subsequently in cell lysis. In addition to this enzymatic process, lysozyme also displays a nonenzymatic mechanism based on its cationic antimicrobial peptide (CAMP) properties, which kills bacteria, most likely through destabilization of the cytoplasmic membrane [9]. In this context, the objective of our study was to investigate the ability of R. equi to resist the muramidase
L. He´bert et al.: Lysozyme Resistance in R. equi
and nonenzymatic mode of action of lysozyme and to evaluate the influence of the virulence plasmid on these capacities.
Materials and Methods Bacterial Strains and Culture Conditions The bacterial strains used in our experiments are the virulence plasmid-bearing strain R. equi ATCC33701 (33701P?) and its isogenic plasmid-cured derivative strain (33701P-) [18]. The presence of the virulence plasmid in 33701P? was verified by PCR detection of the vapA gene [14], to exclude the possibility of plasmid loss (data not shown). Bacteria were grown to stationary phase at 37 °C, in brain–heart infusion (BHI), with vigorous shaking (200 rpm). Preparation of Heat-Inactivated Lysozyme A 100 mg/ml hen egg white lysozyme (Sigma, Ref. L6876) solution was freshly prepared in distilled water (pH 6.5–7), and heat-inactivated lysozyme was prepared by heating in a dry bath for 1 h at 100 °C and was then placed on ice [9]. The effectiveness of the heat-treatment on inactivation of the lysozyme lytic activity has been validated by the help of two previously described Enterococcus faecalis strains [1]: one sensitive (ODSP-pMSP) and one resistant (ODSPpMSP::sigV in the presence of nisin) to the CAMP activity of lysozyme. Lysozyme Challenge Assays
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phosphate buffer 0.1 M pH 7.4 and post-fixed 1 h with 1 % osmium tetroxide in phosphate buffer 0.1 M pH 7.4 (at 4 °C protected from light). The cells were rinsed in phosphate buffer 0.1 M pH 7.4. The cells were pelleted in 1.5 % agar low melting point at 40 °C. The cells were then dehydrated in progressive bath of ethanol (70–100 %) and propylene oxide 100 %, embedded in resin Epon and polymerized 24 h at 60 °C. Ultrathin sections were done and contrasted with uranyl acetate and lead citrate. The cells were observed with transmission electron microscope JEOL 1011 and images were taken with Camera Gatan Orius 200 and digital micrograph software. Scanning Electron Microscope (SEM) Observations Rhodococcus equi cultures were rinsed in PBS (pH 7.2) and fixed with 2.5 % glutaraldehyde in phosphate buffer 0.1 M pH 7.4 overnight at 4 °C. During fixation, the cells were sedimented on ThermanoxÒ coverslip coated with poly-L-lysine. The cells were rinsed in phosphate buffer 0.1 M pH 7.4 and post-fixed 1 h with 1 % osmium tetroxyde in phosphate buffer 0.1 M pH 7.4 (at 4 °C protected from light). The cells were rinsed in phosphate buffer 0.1 M pH 7.4, dehydrated in progressive bath of ethanol (70–100 %) and then critical point dryed (CPD 030 LEICA Microsystem). The cells were sputtered with platinum and observed with scanning electron microscope JEOL 6400F. In Silico Search of Genes Involved in Lysozyme Resistance The search for genes involved in lysozyme resistance was performed by seeking genes known as involved in (i) the modification of PG structure, (ii) the modification of the global charge of the bacterial cell surface, and (iii) the production of lysozyme inhibitors, from the functional annotation content of the virulence plasmid of R. equi 33701P? (GenBank accession number: AP001204) [17], and from genes differentially regulated between R. equi 103S and its isogenic plasmid-cured derivative under virulence plasmid gene-activating conditions (37 °C, pH 6.5) [13].
Cell suspensions were treated by addition of the specified concentration of native or heat-inactivated lysozyme to bacterial cultures grown in stationary phase. Bacterial viability was determined immediately before treatment (control) and after 24 h of incubation at 37 °C, 200 rpm, by viable counts of bacteria performed by plating of tenfold serial dilution in 0.9 % NaCl on BHI agar. Colony-forming units (CFUs) were enumerated after 48 h of incubation at 37 °C by counting two plates at two different dilutions. All experiments were performed in triplicate and statistical analysis of the data was performed using a two-tailed Student’s t test.
Results
Transmission Electron Microscope (TEM) Observations
Analysis of the Survival of R. equi After Exposure to Native or Heat-Inactivated Lysozyme
Rhodococcus equi cultures were rinsed in PBS (pH 7.2), fixed with 2.5 % glutaraldehyde in phosphate buffer 0.1 M pH 7.4 overnight at 4 °C. The cells were rinsed in
We first analyzed the influence of the virulence plasmid on its capacity to contribute to lysozyme resistance of R. equi. For this purpose, we studied the survival capacity of the
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L. He´bert et al.: Lysozyme Resistance in R. equi
also show that the bactericidal capacity of the lysozyme against R. equi largely depends on its muramidase activity. Electron Micrograph of R. equi Following Exposure to Native or Heat-Inactivated Lysozyme
Fig. 1 Susceptibility of R. equi strains 33701P? and 33701P- to native lysozyme or heat-inactivated lysozyme. The virulent R. equi plasmid-bearing strain 33701P? and its isogenic plasmid-cured derivative 33701P- were incubated with catalytic active lysozyme (a), or with heat-inactivated lysozyme (b), added at the specified concentrations. Each value represents the mean CFU/ml (±standard error of mean) from three independent experiments. To facilitate the comparison of 33701P? and 33701P- lysozyme sensitivities, survival rates are reported. Asterisks represent significant differences (*P \ 0.001, **P \ 0.01 as determined by a two-tailed Student’s t test) between the indicated strains
virulence plasmid-bearing R. equi strain 33701P? and its isogenic plasmid-cured derivative strain, 33701P-, under exposure to 0, 1, 5, and 10 mg/ml of native lysozyme or to 0, 10, 25, and 50 mg/ml of heat-inactivated lysozyme (Fig. 1). Our results show clearly that R. equi 33701P? is more resistant to native lysozyme than the strain 33701P-, with survival rates of 61.1, 16.4, and 0.82 for 33701P? and survival rates of 6.8, 0.42, and 0.04 for 33701P-, in the presence of 1, 5, and 10 mg/ml of active lysozyme, respectively. However, the survival capacity of R. equi following exposure to high concentrations of heat-inactivated lysozyme does not seem to depend on the presence of the virulence plasmid, since both strains show a comparable capacity for survival following exposure to 10, 25, and 50 mg/ml of heat-inactivated lysozyme. These results
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SEM was used to examine the effect of either native lysozyme or heat-denatured lysozyme on the morphology of R. equi 33701P? and 33701P- (Fig. 2a). In untreated cultures, we observed a heterogeneous population made up of coccoid and rod-shaped bacteria consistent with the previously described rod–coccus life cycle of R. equi [2]. 24-h treatment of bacteria with 10 mg/ml of native lysozyme resulted in bacterial cell aggregation and the appearance of cellular fragments potentially due to cell lysis by lysozyme. In comparison, 24-h treatment of bacteria with 50 mg/ml of heat-inactivated lysozyme resulted in a higher amount of agglomeration, although the overall morphology of each bacterium did not seem clearly different from the bacteria treated with 10 mg/ml of native lysozyme. No obvious divergence in morphology between 33701P? and 33701Pwas observed under tested conditions. To observe the ultrastructure of the effect of lysozyme on R. equi, we performed TEM observation of 33701P? (Fig. 2b) and 33701P- (data not shown) without treatment or treated with native or heat-inactivated lysozyme. Our results suggest that native and heat-inactivated lysozyme caused the formation of large pores in the bacterial membrane leading to cellular lysis and liberation of intracellular material into the extracellular medium. Moreover, we observed that cells treated with heat-inactivated lysozyme were surrounded by a thick electron-dense layer that we suppose to be the heat-inactivated lysozyme itself, and we could see that the cytoplasm of these cells was more opaque to electron. No obvious divergences between the morphology of 33701P? (Fig. 2b) and 33701P- were observed by TEM (data not shown). In Silico Research of Genes Involved in the Lysozyme Resistance Conferred by the Virulence Plasmid of R. equi Currently, three main mechanisms conferring resistance to lysozyme have been described: (i) the modification of PG structure, (ii) the modification of the global charge of the bacterial cell surface, and (iii) the production of lysozyme inhibitors [12]. To identify the gene(s) involved in the increase of lysozyme resistance capacity of R. equi conferred by the virulence plasmid, we manually screened the gene content of the virulence plasmid of the strains 33701P? [17] and did not identify gene potentially involved in the mechanisms described above. Since the lysozyme resistance conferred by the virulence plasmid
L. He´bert et al.: Lysozyme Resistance in R. equi
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Fig. 2 Electron micrograph observations of R. equi strains 33701P? and 33701P- treated with native lysozyme or heat-inactivated lysozyme. Stationary phase cultures of 33701P? and 33701P- were incubated 24 h with 10 mg/ml of native lysozyme or with 50 mg/ml of heat-inactivated lysozyme or were untreated (negative control).
a SEM observation of 33701P? and 33701P- and b TEM observation of 33701P?. Arrows show potential large pores in the bacterial membrane with liberation of intracellular material into the extracellular medium. Comparable TEM results were obtained with 33701Pthan with 33701P? (data not shown)
may be caused by cross-talk between the plasmid and the chromosome, we screened, in the strain 103S (a strain containing a plasmid virtually identical to the strains 33701P?), for chromosomal genes upregulated in the presence of the virulence plasmid [13]. This analysis did not allow us to identify gene directly involved in lysozyme resistance mechanisms, however, we identified two genes involved in cell wall metabolism and thus potentially in the lysozyme resistance capacity: REQ_05780 which codes for a putative D-ala-D-ala carboxypeptidase, a component potentially involved in PG cross-linking [25], and REQ_09520 which codes for a putative transglycosylase, an enzyme that cleaves PG with the same substrate specificity as lysozyme, leading to the concomitant formation of an intramolecular 1,6-anhydromuramoyl product generally described as involved in PG recycling during the cell cycle [16].
Discussion Numerous levels of lysozyme resistance can be found in different bacteria. As an example, bacteria like Streptococcus pneumoniae R36A [22] and Lactococcus lactis MG1363 [21] have very low levels of lysozyme resistance (below 0.3 mg/ml) whereas other bacteria such as Staphylococcus aureus [9] and E. faecalis [8] are able to resist to lysozyme concentrations as high as 50 mg/ml. Relatively to these observations, our results show that R. equi may be considered as moderately resistant to lysozyme since between 5 and 10 mg/ml of lysozyme is required to reduce viability by 99 %. These levels of resistance to lysozyme seem comparable to the resistance encountered in other Mycolata such as Mycobacterium smegmatis or Mycobacterium tuberculosis which present a minimal inhibitory
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L. He´bert et al.: Lysozyme Resistance in R. equi
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concentration of lysozyme of 2 mg/ml [6] and a survival of 40 % following exposition to 2.5 mg/ml of lysozyme [20], respectively. Moreover, we can specify that the mechanism of action of the lysozyme on R. equi depends on its muramidase activity rather than on its CAMP activity. It was interesting to note that R. equi resistance to the muramidase activity of the lysozyme is increased through the presence of the virulence plasmid. The benefit of enhanced lysozyme resistance seems limited inside macrophages since the virulence plasmid is generally described as conferring R. equi the capacity to inhibit phagosome maturation by preventing their fusion with lysosomes [23], thus limiting the amount of lysozyme in contact with R. equi. However, we can assume that an increased resistance capacity constitutes an advantage enabling survival in the earlier phases of respiratory infection when R. equi interacts with the lysozyme found in tracheobronchial fluids or saliva. SEM and TEM observations did not enable us to identify major morphological divergences between R. equi strains that contained a virulence plasmid and those that did not. In this context, additional biochemical analyses such as determination of the composition of the R. equi cell wall are still needed in order to understand the way in which the virulence plasmid influences the lysozyme resistance capacity of R. equi. Moreover, it should be noted that because of the centrifugation step performed to obtain bacterial concentration during SEM sample preparation, the cell density of each sample is not proportional to the survival rate of R. equi shown in Fig. 1. Cellular fragments resulting from cellular lysis may also have been removed by the centrifugation step. TEM observations suggest that R. equi cell death is due to the creation of a single large pore across the cell membrane, and not to the creation of numerous small pores, leading to an overall destabilization of the membrane. Concerning the opacity of the cytoplasm of cells treated with inactivated lysozyme, we can hypothesize that this phenomenon is due to a different mechanism of action of lysozyme, but more studies are required to confirm this. Research of genes involved in the lysozyme resistance conferred by the virulence plasmid of R. equi allow the identification of two chromosomal genes upregulated in the presence of virulence plasmid, which are potentially involved in the cell wall metabolism of R. equi. This finding suggests the importance of the cross-talk between genes encoded on the chromosome and on the plasmid although implication of these genes in the plasmid-dependant lysozyme resistance of R. equi still needs to be confirmed experimentally by gene disruption or gene overexpression. Although previous studies showed that R. equi’s capacity to resist the effects of acidity [2] and oxidative
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stress [3] is not dependent on the presence of the virulence plasmid, we have demonstrated the participation of the virulence plasmid in R. equi’s stress-resistance mechanisms. Even if several other factors may act in vivo either favoring or limiting lysozyme activity, our finding improves our understanding of the mechanisms potentially enabling R. equi to survive host defenses, and further studies on the cross-talk between the virulence plasmid and the R. equi chromosome will help to characterize these phenomena. Acknowledgments This study was supported by Grants awarded by ANSES, the Regional Council of Basse Normandie and the Ministry of Higher Education, within the framework of Project R25_p3 (CPER 2007–2013). Financial support was obtained from the Institut Franc¸ais du cheval et de l’e´quitation. ANSES’s Dozule´ Laboratory for Equine Diseases is a member of the Hippolia Foundation.
References 1. Benachour A, Ladjouzi R, Le Jeune A, He´bert L, Thorpe S, Courtin P, Chapot-Chartier MP, Prajsnar TK, Foster SJ, Mesnage S (2012) The lysozyme-induced peptidoglycan N-acetylglucosamine deacetylase PgdA (EF1843) is required for Enterococcus faecalis virulence. J Bacteriol 194(22):6066–6073 2. Benoit S, Taouji S, Benachour A, Hartke A (2000) Resistance of Rhodococcus equi to acid pH. Int J Food Microbiol 55(1–3): 295–298 3. Bidaud P, He´bert L, Barbey C, Appourchaux AC, Torelli R, Sanguinetti M, Laugier C, Petry S (2012) Rhodococcus equi’s extreme resistance to hydrogen peroxide is mainly conferred by one of its four catalase genes. PLoS One 7(8):e42396 4. Bruhn O, Grotzinger J, Cascorbi I, Jung S (2011) Antimicrobial peptides and proteins of the horse—insights into a well-armed organism. Vet Res 42(1):98 ˚ , Hammarstro¨m ML 5. Fahlgren A, Hammarstro¨m S, Danielsson A (2003) Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis. Clin Exp Immunol 131(1):90–101 6. Flores AR, Parsons LM, Pavelka MS Jr (2005) Characterization of novel Mycobacterium tuberculosis and Mycobacterium smegmatis mutants hypersusceptible to beta-lactam antibiotics. J Bacteriol 187(6):1892–1900 7. Gigue`re S, Cohen ND, Chaffin MK, Hines SA, Hondalus MK, Prescott JF, Slovis NM (2011) Rhodococcus equi: clinical manifestations, virulence, and immunity. J Vet Intern Med 25(6): 1221–1230 8. He´bert L, Courtin P, Torelli R, Sanguinetti M, Chapot-Chartier M-P, Auffray Y, Benachour A (2007) Enterococcus faecalis constitutes an unusual bacterial model in lysozyme resistance. Infect Immun 75(11):5390–5398 9. Herbert S, Bera A, Nerz C, Kraus D, Peschel A, Goerke C, Meehl M, Cheung A, Go¨tz F (2007) Molecular basis of resistance to muramidase and cationic antimicrobial peptide activity of lysozyme in staphylococci. PLoS Pathog 3(7):e102 10. Hondalus MK, Mosser DM (1994) Survival and replication of Rhodococcus equi in macrophages. Infect Immun 62(10):4167–4175 11. Kedlaya I, Ing MB, Wong SS (2001) Rhodococcus equi infections in immunocompetent hosts: case report and review. Clin Infect Dis 32(3):E39–E46
L. He´bert et al.: Lysozyme Resistance in R. equi 12. Le Jeune A, Torelli R, Sanguinetti M, Giard JC, Hartke A, Auffray Y, Benachour A (2010) The extracytoplasmic function sigma factor SigV plays a key role in the original model of lysozyme resistance and virulence of Enterococcus faecalis. PLoS One 5(3):e9658 13. Letek M, Gonzalez P, MacArthur I, Rodriguez H, Freeman TC, Valero-Rello A, Blanco Mn, Buckley T, Cherevach I, Fahey R, Hapeshi A, Holdstock J, Leadon D, Navas Js, Ocampo A, Quail MA, Sanders M, Scortti MM, Prescott JF, Fogarty U, Meijer WG, Parkhill J, Bentley SD, Va´quez-Boland JA (2010) The genome of a pathogenic Rhodococcus: cooptive virulence underpinned by key gene acquisitions. PLoS Genet 6(9):e1001145 14. Makrai L, Takayama S, De´nes B, Hajto´s I, Sasaki Y, Kakuda T, Tsubaki S, Major A, Fodor L, Varga J, Takai S (2005) Characterization of virulence plasmids and serotyping of Rhodococcus equi isolates from submaxillary lymph nodes of pigs in Hungary. J Clin Microbiol 43(3):1246–1250 15. Perez MGV, Vassilev T, Kemmerly SA (2002) Rhodococcus equi infection in transplant recipients: a case of mistaken identity and review of the literature. Transpl Infect Dis 4(1):52–56 16. Scheurwater E, Reid CW, Clarke AJ (2008) Lytic transglycosylases: bacterial space-making autolysins. Int J Biochem Cell Biol 40(4):586–591 17. Takai S, Hines SA, Sekizaki T, Nicholson VM, Alperin DA, Osaki M, Takamatsu D, Nakamura M, Suzuki K, Ogino N, Kakuda T, Dan H, Prescott JF (2000) DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103. Infect Immun 68(12):6840–6847 18. Takai S, Sekizaki T, Ozawa T, Sugawara T, Watanabe Y, Tsubaki S (1991) Association between a large plasmid and 15- to
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Study of Lysozyme Resistance in Rhodococcus equi Laurent He´bert • Pauline Bidaud • Didier Goux Abdellah Benachour • Claire Laugier • Sandrine Petry
•
Received: 28 June 2013 / Accepted: 17 September 2013 / Published online: 30 October 2013 Ó Springer Science+Business Media New York 2013
Abstract Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.
L. He´bert P. Bidaud C. Laugier S. Petry (&) Dozule´ Laboratory for Equine Diseases, Bacteriology and Parasitology Unit, ANSES, 14430 Goustranville, France e-mail: [email protected] L. He´bert e-mail: [email protected] D. Goux UNICAEN, CMAbio, Normandie Univ, F-14032 Caen, France A. Benachour UNICAEN, U2RM, Normandie Univ, F-14032 Caen, France
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Introduction Rhodococcus equi is a facultative intracellular multihost pathogen affecting mainly foals up to 6 months of age which can cause severe chronic suppurative bronchopneumonia with abscessation, as well as clinical signs of intestinal disease and occasionally septic arthritis or osteomyelitis [7]. In addition, R. equi is an opportunistic zoonotic pathogen commonly identified in immunocompromised people, particularly in patients with AIDS and organ transplant recipients [15, 19], and more rarely in immunocompetent patients [11]. In general, the pathogenicity of R. equi is conferred by the presence of an 80–90 kb virulence plasmid [24] that enables the survival and replication of the bacteria within a membrane-bound vacuole inside macrophages [10]. To survive and colonize a given host, a pathogen must successfully overcome the constitutive or innate defense system and the host’s phagocytic response. One of the most important and widespread components of this defense system is lysozyme. This compound is found in a wide variety of body fluids, such as respiratory and saliva secretions, as well as in cells of the innate immune system, including neutrophils, monocytes, macrophages, and epithelial cells [4, 5]. Lysozyme is an enzyme that cleaves peptidoglycan (PG) between the glycosidic beta-1,4-linked residues of N-acetylmuramic acid and N-acetylglucosamine resulting in the degradation of the PG, and subsequently in cell lysis. In addition to this enzymatic process, lysozyme also displays a nonenzymatic mechanism based on its cationic antimicrobial peptide (CAMP) properties, which kills bacteria, most likely through destabilization of the cytoplasmic membrane [9]. In this context, the objective of our study was to investigate the ability of R. equi to resist the muramidase
L. He´bert et al.: Lysozyme Resistance in R. equi
and nonenzymatic mode of action of lysozyme and to evaluate the influence of the virulence plasmid on these capacities.
Materials and Methods Bacterial Strains and Culture Conditions The bacterial strains used in our experiments are the virulence plasmid-bearing strain R. equi ATCC33701 (33701P?) and its isogenic plasmid-cured derivative strain (33701P-) [18]. The presence of the virulence plasmid in 33701P? was verified by PCR detection of the vapA gene [14], to exclude the possibility of plasmid loss (data not shown). Bacteria were grown to stationary phase at 37 °C, in brain–heart infusion (BHI), with vigorous shaking (200 rpm). Preparation of Heat-Inactivated Lysozyme A 100 mg/ml hen egg white lysozyme (Sigma, Ref. L6876) solution was freshly prepared in distilled water (pH 6.5–7), and heat-inactivated lysozyme was prepared by heating in a dry bath for 1 h at 100 °C and was then placed on ice [9]. The effectiveness of the heat-treatment on inactivation of the lysozyme lytic activity has been validated by the help of two previously described Enterococcus faecalis strains [1]: one sensitive (ODSP-pMSP) and one resistant (ODSPpMSP::sigV in the presence of nisin) to the CAMP activity of lysozyme. Lysozyme Challenge Assays
353
phosphate buffer 0.1 M pH 7.4 and post-fixed 1 h with 1 % osmium tetroxide in phosphate buffer 0.1 M pH 7.4 (at 4 °C protected from light). The cells were rinsed in phosphate buffer 0.1 M pH 7.4. The cells were pelleted in 1.5 % agar low melting point at 40 °C. The cells were then dehydrated in progressive bath of ethanol (70–100 %) and propylene oxide 100 %, embedded in resin Epon and polymerized 24 h at 60 °C. Ultrathin sections were done and contrasted with uranyl acetate and lead citrate. The cells were observed with transmission electron microscope JEOL 1011 and images were taken with Camera Gatan Orius 200 and digital micrograph software. Scanning Electron Microscope (SEM) Observations Rhodococcus equi cultures were rinsed in PBS (pH 7.2) and fixed with 2.5 % glutaraldehyde in phosphate buffer 0.1 M pH 7.4 overnight at 4 °C. During fixation, the cells were sedimented on ThermanoxÒ coverslip coated with poly-L-lysine. The cells were rinsed in phosphate buffer 0.1 M pH 7.4 and post-fixed 1 h with 1 % osmium tetroxyde in phosphate buffer 0.1 M pH 7.4 (at 4 °C protected from light). The cells were rinsed in phosphate buffer 0.1 M pH 7.4, dehydrated in progressive bath of ethanol (70–100 %) and then critical point dryed (CPD 030 LEICA Microsystem). The cells were sputtered with platinum and observed with scanning electron microscope JEOL 6400F. In Silico Search of Genes Involved in Lysozyme Resistance The search for genes involved in lysozyme resistance was performed by seeking genes known as involved in (i) the modification of PG structure, (ii) the modification of the global charge of the bacterial cell surface, and (iii) the production of lysozyme inhibitors, from the functional annotation content of the virulence plasmid of R. equi 33701P? (GenBank accession number: AP001204) [17], and from genes differentially regulated between R. equi 103S and its isogenic plasmid-cured derivative under virulence plasmid gene-activating conditions (37 °C, pH 6.5) [13].
Cell suspensions were treated by addition of the specified concentration of native or heat-inactivated lysozyme to bacterial cultures grown in stationary phase. Bacterial viability was determined immediately before treatment (control) and after 24 h of incubation at 37 °C, 200 rpm, by viable counts of bacteria performed by plating of tenfold serial dilution in 0.9 % NaCl on BHI agar. Colony-forming units (CFUs) were enumerated after 48 h of incubation at 37 °C by counting two plates at two different dilutions. All experiments were performed in triplicate and statistical analysis of the data was performed using a two-tailed Student’s t test.
Results
Transmission Electron Microscope (TEM) Observations
Analysis of the Survival of R. equi After Exposure to Native or Heat-Inactivated Lysozyme
Rhodococcus equi cultures were rinsed in PBS (pH 7.2), fixed with 2.5 % glutaraldehyde in phosphate buffer 0.1 M pH 7.4 overnight at 4 °C. The cells were rinsed in
We first analyzed the influence of the virulence plasmid on its capacity to contribute to lysozyme resistance of R. equi. For this purpose, we studied the survival capacity of the
123
354
L. He´bert et al.: Lysozyme Resistance in R. equi
also show that the bactericidal capacity of the lysozyme against R. equi largely depends on its muramidase activity. Electron Micrograph of R. equi Following Exposure to Native or Heat-Inactivated Lysozyme
Fig. 1 Susceptibility of R. equi strains 33701P? and 33701P- to native lysozyme or heat-inactivated lysozyme. The virulent R. equi plasmid-bearing strain 33701P? and its isogenic plasmid-cured derivative 33701P- were incubated with catalytic active lysozyme (a), or with heat-inactivated lysozyme (b), added at the specified concentrations. Each value represents the mean CFU/ml (±standard error of mean) from three independent experiments. To facilitate the comparison of 33701P? and 33701P- lysozyme sensitivities, survival rates are reported. Asterisks represent significant differences (*P \ 0.001, **P \ 0.01 as determined by a two-tailed Student’s t test) between the indicated strains
virulence plasmid-bearing R. equi strain 33701P? and its isogenic plasmid-cured derivative strain, 33701P-, under exposure to 0, 1, 5, and 10 mg/ml of native lysozyme or to 0, 10, 25, and 50 mg/ml of heat-inactivated lysozyme (Fig. 1). Our results show clearly that R. equi 33701P? is more resistant to native lysozyme than the strain 33701P-, with survival rates of 61.1, 16.4, and 0.82 for 33701P? and survival rates of 6.8, 0.42, and 0.04 for 33701P-, in the presence of 1, 5, and 10 mg/ml of active lysozyme, respectively. However, the survival capacity of R. equi following exposure to high concentrations of heat-inactivated lysozyme does not seem to depend on the presence of the virulence plasmid, since both strains show a comparable capacity for survival following exposure to 10, 25, and 50 mg/ml of heat-inactivated lysozyme. These results
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SEM was used to examine the effect of either native lysozyme or heat-denatured lysozyme on the morphology of R. equi 33701P? and 33701P- (Fig. 2a). In untreated cultures, we observed a heterogeneous population made up of coccoid and rod-shaped bacteria consistent with the previously described rod–coccus life cycle of R. equi [2]. 24-h treatment of bacteria with 10 mg/ml of native lysozyme resulted in bacterial cell aggregation and the appearance of cellular fragments potentially due to cell lysis by lysozyme. In comparison, 24-h treatment of bacteria with 50 mg/ml of heat-inactivated lysozyme resulted in a higher amount of agglomeration, although the overall morphology of each bacterium did not seem clearly different from the bacteria treated with 10 mg/ml of native lysozyme. No obvious divergence in morphology between 33701P? and 33701Pwas observed under tested conditions. To observe the ultrastructure of the effect of lysozyme on R. equi, we performed TEM observation of 33701P? (Fig. 2b) and 33701P- (data not shown) without treatment or treated with native or heat-inactivated lysozyme. Our results suggest that native and heat-inactivated lysozyme caused the formation of large pores in the bacterial membrane leading to cellular lysis and liberation of intracellular material into the extracellular medium. Moreover, we observed that cells treated with heat-inactivated lysozyme were surrounded by a thick electron-dense layer that we suppose to be the heat-inactivated lysozyme itself, and we could see that the cytoplasm of these cells was more opaque to electron. No obvious divergences between the morphology of 33701P? (Fig. 2b) and 33701P- were observed by TEM (data not shown). In Silico Research of Genes Involved in the Lysozyme Resistance Conferred by the Virulence Plasmid of R. equi Currently, three main mechanisms conferring resistance to lysozyme have been described: (i) the modification of PG structure, (ii) the modification of the global charge of the bacterial cell surface, and (iii) the production of lysozyme inhibitors [12]. To identify the gene(s) involved in the increase of lysozyme resistance capacity of R. equi conferred by the virulence plasmid, we manually screened the gene content of the virulence plasmid of the strains 33701P? [17] and did not identify gene potentially involved in the mechanisms described above. Since the lysozyme resistance conferred by the virulence plasmid
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Fig. 2 Electron micrograph observations of R. equi strains 33701P? and 33701P- treated with native lysozyme or heat-inactivated lysozyme. Stationary phase cultures of 33701P? and 33701P- were incubated 24 h with 10 mg/ml of native lysozyme or with 50 mg/ml of heat-inactivated lysozyme or were untreated (negative control).
a SEM observation of 33701P? and 33701P- and b TEM observation of 33701P?. Arrows show potential large pores in the bacterial membrane with liberation of intracellular material into the extracellular medium. Comparable TEM results were obtained with 33701Pthan with 33701P? (data not shown)
may be caused by cross-talk between the plasmid and the chromosome, we screened, in the strain 103S (a strain containing a plasmid virtually identical to the strains 33701P?), for chromosomal genes upregulated in the presence of the virulence plasmid [13]. This analysis did not allow us to identify gene directly involved in lysozyme resistance mechanisms, however, we identified two genes involved in cell wall metabolism and thus potentially in the lysozyme resistance capacity: REQ_05780 which codes for a putative D-ala-D-ala carboxypeptidase, a component potentially involved in PG cross-linking [25], and REQ_09520 which codes for a putative transglycosylase, an enzyme that cleaves PG with the same substrate specificity as lysozyme, leading to the concomitant formation of an intramolecular 1,6-anhydromuramoyl product generally described as involved in PG recycling during the cell cycle [16].
Discussion Numerous levels of lysozyme resistance can be found in different bacteria. As an example, bacteria like Streptococcus pneumoniae R36A [22] and Lactococcus lactis MG1363 [21] have very low levels of lysozyme resistance (below 0.3 mg/ml) whereas other bacteria such as Staphylococcus aureus [9] and E. faecalis [8] are able to resist to lysozyme concentrations as high as 50 mg/ml. Relatively to these observations, our results show that R. equi may be considered as moderately resistant to lysozyme since between 5 and 10 mg/ml of lysozyme is required to reduce viability by 99 %. These levels of resistance to lysozyme seem comparable to the resistance encountered in other Mycolata such as Mycobacterium smegmatis or Mycobacterium tuberculosis which present a minimal inhibitory
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concentration of lysozyme of 2 mg/ml [6] and a survival of 40 % following exposition to 2.5 mg/ml of lysozyme [20], respectively. Moreover, we can specify that the mechanism of action of the lysozyme on R. equi depends on its muramidase activity rather than on its CAMP activity. It was interesting to note that R. equi resistance to the muramidase activity of the lysozyme is increased through the presence of the virulence plasmid. The benefit of enhanced lysozyme resistance seems limited inside macrophages since the virulence plasmid is generally described as conferring R. equi the capacity to inhibit phagosome maturation by preventing their fusion with lysosomes [23], thus limiting the amount of lysozyme in contact with R. equi. However, we can assume that an increased resistance capacity constitutes an advantage enabling survival in the earlier phases of respiratory infection when R. equi interacts with the lysozyme found in tracheobronchial fluids or saliva. SEM and TEM observations did not enable us to identify major morphological divergences between R. equi strains that contained a virulence plasmid and those that did not. In this context, additional biochemical analyses such as determination of the composition of the R. equi cell wall are still needed in order to understand the way in which the virulence plasmid influences the lysozyme resistance capacity of R. equi. Moreover, it should be noted that because of the centrifugation step performed to obtain bacterial concentration during SEM sample preparation, the cell density of each sample is not proportional to the survival rate of R. equi shown in Fig. 1. Cellular fragments resulting from cellular lysis may also have been removed by the centrifugation step. TEM observations suggest that R. equi cell death is due to the creation of a single large pore across the cell membrane, and not to the creation of numerous small pores, leading to an overall destabilization of the membrane. Concerning the opacity of the cytoplasm of cells treated with inactivated lysozyme, we can hypothesize that this phenomenon is due to a different mechanism of action of lysozyme, but more studies are required to confirm this. Research of genes involved in the lysozyme resistance conferred by the virulence plasmid of R. equi allow the identification of two chromosomal genes upregulated in the presence of virulence plasmid, which are potentially involved in the cell wall metabolism of R. equi. This finding suggests the importance of the cross-talk between genes encoded on the chromosome and on the plasmid although implication of these genes in the plasmid-dependant lysozyme resistance of R. equi still needs to be confirmed experimentally by gene disruption or gene overexpression. Although previous studies showed that R. equi’s capacity to resist the effects of acidity [2] and oxidative
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stress [3] is not dependent on the presence of the virulence plasmid, we have demonstrated the participation of the virulence plasmid in R. equi’s stress-resistance mechanisms. Even if several other factors may act in vivo either favoring or limiting lysozyme activity, our finding improves our understanding of the mechanisms potentially enabling R. equi to survive host defenses, and further studies on the cross-talk between the virulence plasmid and the R. equi chromosome will help to characterize these phenomena. Acknowledgments This study was supported by Grants awarded by ANSES, the Regional Council of Basse Normandie and the Ministry of Higher Education, within the framework of Project R25_p3 (CPER 2007–2013). Financial support was obtained from the Institut Franc¸ais du cheval et de l’e´quitation. ANSES’s Dozule´ Laboratory for Equine Diseases is a member of the Hippolia Foundation.
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