Microbial Pathogenesis 1992 ; 13: 3 1 7-323
Mutant of Salmonella typhimurium lacking the inhibitory function for phagosome-lysosome fusion in murine macrophages Yoshio Ishibashi, Kan Nobuta and Toshihiko Arai Department of Microbiology, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo 154, Japan
(Received February 24, 1992 ; accepted in revised form August 3, 1992)
Ishibashi, Y . (Dept of Microbiology, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo 154, Japan), K . Nobuta and Toshihiko Arai . Mutant of Salmonella typhimurium lacking the inhibitory function for phagosome-lysosome fusion in murine macrophages . Microbial Pathogenesis 1992 ; 13 : 317-323 . It has recently been described that Salmonella typhimurium is capable of inhibiting phagosome-lysosome fusion in murine macrophages after ingestion . We selected a mutant of S . typhimurium lacking the phagosome-lysosome fusion inhibitory function from a collection of Tn5-insertion mutants and examined its relevance to the pathogenesis in mice . The Tn5insertion mutant which has a defect in fusion inhibitory function was found to be significantly sensitive to the intracellular killing by murine macrophages in vitro . However, the loss of the fusion inhibitory function did not reduce the level of virulence for mice in vivo . These results demonstrated that fusion inhibition did not play a critical role in the pathogenesis of S . typhimurium although it might contribute to at least a part of the resistance against macrophage killing mechanisms . Key words : phagosome-lysosome fusion ; macrophage ; transposon Tn5 ; Salmonella typhimurium .
Introduction Salmonella typhimurium infection causes a typhoid fever-like disease in mice and is used as an animal model of human typhoid fever .' It has been well documented that S . typhimurium is a facultative intracellular pathogen which can survive within murine macrophages,' although there has been a controversial viewpoint that S . typhimurium is killed by murine phagocytes but readily propagates in extracellular locations of liver and spleen . 34 We previously demonstrated that S . typhimurium was able to inhibit phagosomelysosome fusion in murine macrophages, suggesting that the fusion inhibitory function might be involved in the pathogenesis of S . typhimurium . 5 Buchmeier et al.' has also confirmed the inhibition of phagosome-lysosome fusion by S. typhimurium from electron microscopic observations . Similarly, several reports have postulated that phagosome-lysosome fusion inhibition may be involved in the pathogenesis of Mycobacterium tuberculosis,' Mycobacterium microti' and Toxoplasma gondii. 9 H ow ever, there has been no direct evidence to prove the relationship between the fusion inhibitory function and the virulence of these pathogens as well in S . typhimurium . Transposon mutagenesis can be a powerful tool for analysing virulence determinants 0882-4010/92/100317+07 $08 .00/0
© 1992 Academic Press Limited
Y . Ishibashi et al .
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of pathogenic bacteria . 10-13 In an attempt to explore the role of the fusion inhibitory function in pathogenesis of S. typhimurium, we constructed a bank of Tn5 insertion mutants from a virulent strain of S . typhimurium, and selected the mutant strain that has a defect in the fusion inhibitory function . The mutant strain obtained was characterized to examine the contribution of the fusion inhibitory function to the virulence of S. tgphimurium .
Results Selection and characterization of mutant strain that has a defect in the fusion inhibitory function Tn5 insertion mutants were screened for loss of the phagosome-lysosome fusion inhibitory function of S . typhimurium . Among 2500 mutant strains of S .-typhimurium 5170, a mutant 5635 was found to be defective in the inhibitory function for phagosome-lysosome fusion after ingestion by macrophages (Table 1) . S. typhimurium 5170, the parental strain, markedly impaired phagosome-lysosome fusion in murine macrophages, whereas mutant 5635 was capable of inducing efficient phagosome-lysosome fusion in macrophages . There was no difference in the phagocytic activities between the parental strain and the mutantinfected macrophages . Figure 1 shows the typical microphotographs of phagosomelysosome fusion in macrophages infected with the parental strain or the mutant . The phagosomes containing 5170 appeared as dark spaces against a background of fluorescent lysosomes, indicating the absence of fusion [Fig . 1 (A)] . Phagolysosome formation was recognized by transfer of sulphorhodamine into the phagosomes containing 5635 [Fig . 1 (B)] . Further characterization of the mutant was performed . Both parental strain and 5635 reacted equally well with H-i and 0-4 antisera . Both wild-type and 5635 were found to possess complete, smooth lipopolysaccharide (LPS) with no observable differences as judged by SDS-PAGE for LPS (data not shown) . The Tn5 mutant was able to grow on minimal medium containing glucose, indicating that auxotrophic requirements were not developed during the construction of the strain . The mutant was highly motile as determined by stabbing into semi-solid medium . Virulence of the Tn 5 mutant lacking the fusion inhibitory function To investigate the contribution of the fusion inhibitory function to the virulence of S. typhimurium, the resistance of the mutant against intracellular killing by macrophages was examined in vitro (Fig . 2) . The number of viable bacteria of the parental strain within macrophages remained unchanged for up to 48 h although a slight decrease in
Table 1 Phagocytosis and subsequent phagosomelysosome fusion in murine macrophages infected with Tn5 mutant of S. typhimurium Strain 5170 (wild-type) 5635 (5170 : :Tn5)
Phagocytic index
Fusion index
218 .6±29 .0 247 .2±20 .4
4 .51+0 .50 54 .63±9 .06
Sulphorhodamine-labelled macrophages (1 x 10 5 cells/well) were infected with bacteria at a ratio of 1 :5 in the presence of 10% normal fresh serum . After 1 h incubation, phagocytosis and phagosomelysosome fusion were assayed . The values represent the means±SE of four experiments .
Pathogenesis of S. typhimurium
31 9
A
B
Fig . 1 . Fluorescence micrographs of sulphorhodamine-stained macrophages . (a) Macrophage prelabelled with sulphorhodamine were incubated with S . typhimurium 5170 at 37 ° C for 60 min . Macrophages contained many sulphorhodamine-stained lysosomes surrounding dark spaces (arrowhead) which were unstained bacteria in non-fused phagosomes (X900) . (b) Macrophages were infected with S . typhimurium 5635 in the same manner . A number of bacteria were stained with sulphorhodamine (arrow), indicating the diffusion of the lysosome marker dye into phagosomes (x 900) .
or 5635 (0) at a
32 0
Y . Ishibashi et al.
4
• •
3
2 •
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48 24 Time after infection (h) Fig . 2 . Intracellular killing of Tn5 mutant of S . typhimurium by cultured murine macrophages in vitro . Macrophage monolayers (5x105 cells/well) were infected with S . typhimurium 5170 ( •) ratio of 1 :1 in the presence of 10% fresh serum . After 1 h incubation, infected macrophages were washed with RPM 11640 containing 5 pg/ml of gentamicin to remove the extracellular bacteria and further incubated at 37°C in 5% C02 . At various time intervals, the number of viable bacteria in macrophages were determined by colony counting after cell lysis with PBS containing 0 .05% triton x-100 . The values represent the mean±SE of five experiments . 1
6
the first 6 h was observed . In contrast, the viable number of the mutant strain within macrophages continued to decline over the entire 48 h infection period . We also examined the virulence of the mutant in mice in vivo . Mice were infected intraperitoneally (i .p .) or orally with a serial dilution of S . typhimurium 5635, and then 50% lethal dose (LD50) values were determined after 3 weeks . As shown in Table 2, LD50 values of the mutant strain were almost similar to those of parental strain . Mice infected by the same route displayed similar average times to death regardless of the challenge strain . No remarkable difference in the symptom was observed between parent strain and the mutant-infected mice . The possibility that the mutant strain reverted to wild-type in vivo was examined in parallel with the LD50 determinations . Additional three mice were challenged i .p . with 102 cfu of 5635 strain and then killed at 5 days after challenge . Their spleens were homogenized, appropriately diluted with saline and plated on L-agar with or without
Table 2 Virulence of Tn5 mutant of S . typhimurium to mice in vivo i .p .
P.O .
Strain
LD50
Average time a to death (day)
LD50
Average time to death (day)
5170 (wild-type) 5635 (5170 : :Tn5)
70 30
7 8
1 .2 x 105 9 .0x10°
11 11
Mice were infected intraperitoneally (i .p .) or orally (P .O .) with a serial dilution of S. typhimurium, and then LD50 values were determined after 3 weeks . The values represent the means of two experiments . Data taken 3 weeks post-challenge for mice receiving approximately 1 LD50 .
Pathogenesis of S. typhimurium
321
kanamycin (25,ug/ml) . Viable counts were found to be almost the same regardless of the presence of kanamycin, indicating that the mutant strain 5635 is still carrying the transposon insertion .
Discussion Our previous report has shown that S. typhimurium is capable of inhibiting phagosomelysosome fusion in murine macrophages after ingestion, suggesting that the fusion inhibitory function may be a virulence factor of S . typhimurium . To find an answer, we now screened a mutant lacking the fusion inhibitory function from a bank of Tn5 insertion mutants of S. typhimurium 5170 . Among 2500 mutants, only one mutant was found to be defective in the fusion inhibitory function . The mutant 5635 had no change in LPS composition, mobility and auxotrophic requirement . It has been reported that the virulence-associated plasmids in Salmonellae encode several virulence factors ."-" In our study, the transposon insertion appeared not to be in the plasmid but in the chromosome as determined by Southern hybridization using Tn5fragment as a probe (data not shown) . Horwitz" has previously demonstrated that the mutant strains of Legione//a pneumophila lacking phagosome-lysosome fusion inhibitory function are able to survive but not grow within phagocytes, and that these mutant strains are avirulent in vivo . Our study found that mutant 5635 was significantly sensitive to the intracellular killing by cultured macrophages as compared with the parental strain . However, the loss of the fusion inhibitory function was not associated with reduction of virulence in mice in vivo . These findings demonstrated that the fusion inhibitory function might not play a critical role for the net virulence of S . typhimurium, although it might contribute to at least a part of the intracellular survival within macrophages . Virulence of S. typhimurium has been attributed to various factors, 18 including resistance to reactive oxygen metabolites 19 and resistance to bactericidal peptides such as defensin . 20 Although the relative contribution of these mechanisms to virulence of S . typhimurium is far from being fully understood, it seemed likely that total pathogenesis of S . typhimurium might result from the complex of multiple virulence factors rather than a single virulence factor. An alternative interpretation might be possible . Lin et a/. 4 have demonstrated that the virulent strain of S . typhimurium is easily killed by phagocytes, but can efficiently grow in extracellular locations . It therefore seemed likely that an efficient extracellular growth of S . typhimurium in vivo might overcome the intracellular killing by macrophages even if the mutant strain had a defect in the fusion inhibitory function . Further analysis of the gene and its product(s) with regard to the fusion inhibitory function may provide an important information for understanding the interaction between S . typhimurium and host macrophages at the molecular level .
Materials and methods
Animals. Male BALB/c mice, aged 6 weeks, were used . Bacteria . Salmonella typhimurium 5170 wild-type, an isolate from a field case of calf enteritis, was kindly provided by Dr N . Terakado of the National Institute of Animal Health . It was grown in Luria broth (LB) at 37°C and harvested during exponential growth . Bacterial concentrations were determined by measuring the turbidity at 660 nm' and adjusted to desired concentration in Dulbecco's phosphate buffered saline (PBS) . Isolation of Tn5 mutants . Tn5 insertions were made by mating a temperature-sensitive
322
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R388ts : :Tn5 into 5170 at 32 ° C and selecting trimetprim-sensitive and kanamycin-resistant derivatives on Luria plates at 42°C as described previously . 21 Isolated mutants were cultured with LB or Luria agar plates in the presence of kanamycin (25 pg/ml) to prevent the accumulation of revertants . Reagents and media . RPM11640 medium and Dulbecco's PBS were obtained from Nissui Seiyaku Co ., Ltd, Tokyo, Japan . Gentamicin was purchased from Dainippon Seiyaku Co ., Ltd, Osaka, Japan . Sulphorhodamine 101 was purchased from Sigma Chemical Co ., St Louis, MO, U .S .A . Preparation of macrophages . Resident peritoneal macrophages were obtained by washing the peritoneal cavity with RPMI1640 containing 5 pg/ml of gentamicin (GM) and 5 U/ml of heparin as described previously .' To obtain macrophage monolayers, 100 PI of the macrophage suspensions (1 x106 cells/ml) were plated on eight-well HTC slides (Cel-Line Inc ., Newfield, NJ) and incubated for 1 h at 37°C . After incubation, macrophage monolayers (1 x 105 cells/well) were rinsed and cultured with RPMI1640-GM containing 10% foetal bovine serum (FBS) for 2 days at 37°C in 5% CO 2 before use . For intracellular killing assay, the macrophage monolayers (5 x 10 5 cells/well) were prepared in 24-well culture dishes in the same way . Assay for phagosome-lysosome fusion and phagocytosis . Phagosome-lysosome fusion in macrophages was assessed by using fluorescent vital dyes as described previously . 5.22 In brief, sulphorhodamine was used as a fluorescent marker for monitoring phagosome-lysosome fusion .' Macrophage monolayers were labelled with 200 Mg/ml of sulphorhodamine in RPMI1640 overnight at 37°C, and then rinsed with RPM11640 . Macrophages were infected with bacteria (macrophage to bacteria ratio of 1 :5) in a total volume of 100 pl of RMPI1640 containing 10% normal fresh serum . After incubation, macrophages were washed three times with RPM 11640GM and allowed to air dry, followed by UV-irradiation for 5 min to kill the bacteria . The activity of phagosome-lysosome fusion was determined by observing sulphorhodamine-stained bacteria under fluorescence microscope . Subsequently, macrophage monolayers were fixed with 4% paraformaldehyde, stained with Giemsa, and then phagocytic activity was determined by observing intracellular bacteria by light microscope . The fusion index and the phagocytic index were defined as the percentage positive fusion multiplied by the average number of fused phagosome per cell and the percentage positive phagocytosis multiplied by the average number of phagocytized bacteria per cell, respectively .' Intracellular killing by macrophages . Intracellular killing of bacteria by murine macrophages was assessed according to the method of Lissner 23 with a slight modification . The minimum inhibitory concentrations (MICs) of gentamicin for S . typhimurium strains were determined in RPMI1640 medium containing 10% FBS as described previously ." The MIC of gentamicin for both S. typhimurium 5170 and Tn5 insertion mutant was 2 .5 pg/ml . Thus, the concentration of 5 .0 pg/ml of gentamicin was used for the prevention of extracellular growth of bacteria . Macrophage monolayers (5x 10 5 cells/well) were infected with bacteria at a ratio of 1 :1 in the presence of 10% fresh serum . After 1 h incubation, infected macrophages were washed with RPM 11640 containing 5 pg/ml of gentamicin to remove the extracellular bacteria, and further incubated at 37°C in 5% CO 2 . After incubation, the number of viable bacteria in macrophages were determined by colony counting after cell lysis by PBS containing 0 .05% triton x-1 00 . Determination of LD 50 values. Animal inoculations for the determination of the LD 50 values were performed as described previously ." Five mice were housed in a cage . For intraperitoneal inoculation, mice were infected i .p . with 0 .2 ml of bacterial suspension serially diluted in buffered saline containing 0 .1% gelatin, and then LD 50 values were determined after 3 weeks . For oral challenge, mice were starved for food and water for 6 h and then fed 50 pl of 10% sodium bicarbonate, followed by 20 pl of bacterial suspension . LD 50 titers were calculated by the method of Reed and Muench 21 from results obtained from five mice per inoculum dose .
References 1 . Lissner CR, Weinstein DL, O'Brien AD . Mouse chromosome 1 Ity locus regulates microbicidal activity of isolated peritoneal macrophages against a diverse group of intracellular and extracellular bacteria . J Immunol 1985; 135 :544-7 .
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2 . Mackaness GB, Blanden RV, Collins FM . Host-parasite relations in mouse typhoid . J Exp Med 1966 ; 124 :573-83 . 3 . Hsu HS . Pathogenesis and immunity in murine salmonellosis . Microbiol Rev 1989 ; 53 : 390-409 . 4 . Lin FR, Hsu HS, Mumaw VR, Nakoneczna I . Intracellular destruction of salmonellae in genetically resistant mice. J Med Microbiol 1989 ; 30 : 79-87 . 5 . Ishibashi Y, Arai T . Specific inhibition of phagosome-lysosome fusion in murine macrophages mediated by Salmonella typhimurium infection . FEMS Microbiol Immunol 1990; 64 :35-44 . 6 . Buchmeier NA, Heffron F. Inhibition of phagosome-lysosome fusion by Salmonella typhimurium . Infect Immun 1991 ; 59 : 2232-8 . 7 . Armstrong JA, Hart PD . Response of cultured macrophages to Mycobacterium tuberculosis with observations on fusion of lysosomes with phagosomes . J Exp Med 1971 ; 134 : 717-40 . 8 . Lowrie DB, Jackett PS, Ratcliffe NA . Mycobacterium microti may protect itself from intracellular destruction by releasing cyclic AMP into phagosomes . Nature 1975 ; 254 : 600-2 . 9 . Jones TC, Hirsch JG . The interaction between Toxoplasma gondii and mammalian cells . II . The absence of lysosomal fusion with phagocytic vacuoles containing living parasites . J Exp Med 1972 ; 136 : 117394 . 10 . Gaillard JL, Berche P, Sansonetti P . Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect Immun 1986 ; 52 : 50-5 . 11 . Kress Y, Bloom BR, Witter M, Rowen A, Tanowitz H . Resistance of Trypanosoma crusi to killing by macrophages . Nature 1975 ; 257 : 394-6 . 12 . Lockman HA, Curtiss R Ill . Salmonella typhimurium mutants lacking flagella or motility remain virulent in BALB/c mice . Infect Immun 1990 ; 58 :137-43 . 13 . Fields PI, Swanson RV, Haidaris CG, Heffron F . Mutants of Salmonella typhimurium that can survive within the macrophage are avirulent . Proc Natl Acad Sci USA 1986 ; 83 : 5189-93 . 14 . Hackett J, Wyk P, Reeves P, Mathan V . Mediation of serum resistance on Salmonella typhimurium by an 11 -kilodalton polypeptide encoded by the cryptic plasmid . J Infect Dis 1987 ; 155 : 540-9 . 15 . Pardon P, Popoff MY, Coynault C, Marly J, Miras I . Virulence-associated plasmids of Salmonella serotype typhimurium in experimental murine infection . Ann Microbiol (Paris) 1896 ; 13713 : 47-60 . 16 . Gulig PA, Curtiss R III . Plasmid-associated virulence of Salmonella typhimurium . Infect Immun 1987 ; 55 :2891-901 . 17 . Horwitz MA . Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes . J Exp Med 1987 ; 166 :1310-28 . 18 . Miller SI . PhoP/PhoQ : macrophage-specific modulators of Salmonella virulence? Mol Microbiol 1991 ; 5 :2073-8. 19 . Ishibashi Y, Arai T . Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites . FEMS Microbiol Immunol 1989 ; 47 : 279-84 . 20 . Fields PI, Groisman EA, Heffron F . A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells . Science 1989 ; 243 : 1059-62 . 21 . Sasakawa C, Yoshikawa M . A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis . Gene 1987 ; 56 : 283-8 . 22 . Goren MB, Swendsen CL, Fiscus J, Miranti C . Fluorescent markers for studying phagosome-lysosome fusion . J Leukocyte Biol 1984; 36 : 273-92 . 23 . Lissner CR, Swanson RN, O'Brien AD . Genetic control of the innate resistance of mice to Salmonella typhimurium : expression of the lty gene in peritoneal and splenic macrophages isolated in vitro . J Immunol 1983 ; 131 : 3006-13 . 24 . Reed LJ, Muench H . A simple method of estimating fifty percent endpoints. Am J Hyg 1938 ; 27 : 493-7 .
Mutant of Salmonella typhimurium lacking the inhibitory function for phagosome-lysosome fusion in murine macrophages Yoshio Ishibashi, Kan Nobuta and Toshihiko Arai Department of Microbiology, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo 154, Japan
(Received February 24, 1992 ; accepted in revised form August 3, 1992)
Ishibashi, Y . (Dept of Microbiology, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo 154, Japan), K . Nobuta and Toshihiko Arai . Mutant of Salmonella typhimurium lacking the inhibitory function for phagosome-lysosome fusion in murine macrophages . Microbial Pathogenesis 1992 ; 13 : 317-323 . It has recently been described that Salmonella typhimurium is capable of inhibiting phagosome-lysosome fusion in murine macrophages after ingestion . We selected a mutant of S . typhimurium lacking the phagosome-lysosome fusion inhibitory function from a collection of Tn5-insertion mutants and examined its relevance to the pathogenesis in mice . The Tn5insertion mutant which has a defect in fusion inhibitory function was found to be significantly sensitive to the intracellular killing by murine macrophages in vitro . However, the loss of the fusion inhibitory function did not reduce the level of virulence for mice in vivo . These results demonstrated that fusion inhibition did not play a critical role in the pathogenesis of S . typhimurium although it might contribute to at least a part of the resistance against macrophage killing mechanisms . Key words : phagosome-lysosome fusion ; macrophage ; transposon Tn5 ; Salmonella typhimurium .
Introduction Salmonella typhimurium infection causes a typhoid fever-like disease in mice and is used as an animal model of human typhoid fever .' It has been well documented that S . typhimurium is a facultative intracellular pathogen which can survive within murine macrophages,' although there has been a controversial viewpoint that S . typhimurium is killed by murine phagocytes but readily propagates in extracellular locations of liver and spleen . 34 We previously demonstrated that S . typhimurium was able to inhibit phagosomelysosome fusion in murine macrophages, suggesting that the fusion inhibitory function might be involved in the pathogenesis of S . typhimurium . 5 Buchmeier et al.' has also confirmed the inhibition of phagosome-lysosome fusion by S. typhimurium from electron microscopic observations . Similarly, several reports have postulated that phagosome-lysosome fusion inhibition may be involved in the pathogenesis of Mycobacterium tuberculosis,' Mycobacterium microti' and Toxoplasma gondii. 9 H ow ever, there has been no direct evidence to prove the relationship between the fusion inhibitory function and the virulence of these pathogens as well in S . typhimurium . Transposon mutagenesis can be a powerful tool for analysing virulence determinants 0882-4010/92/100317+07 $08 .00/0
© 1992 Academic Press Limited
Y . Ishibashi et al .
31 8
of pathogenic bacteria . 10-13 In an attempt to explore the role of the fusion inhibitory function in pathogenesis of S. typhimurium, we constructed a bank of Tn5 insertion mutants from a virulent strain of S . typhimurium, and selected the mutant strain that has a defect in the fusion inhibitory function . The mutant strain obtained was characterized to examine the contribution of the fusion inhibitory function to the virulence of S. tgphimurium .
Results Selection and characterization of mutant strain that has a defect in the fusion inhibitory function Tn5 insertion mutants were screened for loss of the phagosome-lysosome fusion inhibitory function of S . typhimurium . Among 2500 mutant strains of S .-typhimurium 5170, a mutant 5635 was found to be defective in the inhibitory function for phagosome-lysosome fusion after ingestion by macrophages (Table 1) . S. typhimurium 5170, the parental strain, markedly impaired phagosome-lysosome fusion in murine macrophages, whereas mutant 5635 was capable of inducing efficient phagosome-lysosome fusion in macrophages . There was no difference in the phagocytic activities between the parental strain and the mutantinfected macrophages . Figure 1 shows the typical microphotographs of phagosomelysosome fusion in macrophages infected with the parental strain or the mutant . The phagosomes containing 5170 appeared as dark spaces against a background of fluorescent lysosomes, indicating the absence of fusion [Fig . 1 (A)] . Phagolysosome formation was recognized by transfer of sulphorhodamine into the phagosomes containing 5635 [Fig . 1 (B)] . Further characterization of the mutant was performed . Both parental strain and 5635 reacted equally well with H-i and 0-4 antisera . Both wild-type and 5635 were found to possess complete, smooth lipopolysaccharide (LPS) with no observable differences as judged by SDS-PAGE for LPS (data not shown) . The Tn5 mutant was able to grow on minimal medium containing glucose, indicating that auxotrophic requirements were not developed during the construction of the strain . The mutant was highly motile as determined by stabbing into semi-solid medium . Virulence of the Tn 5 mutant lacking the fusion inhibitory function To investigate the contribution of the fusion inhibitory function to the virulence of S. typhimurium, the resistance of the mutant against intracellular killing by macrophages was examined in vitro (Fig . 2) . The number of viable bacteria of the parental strain within macrophages remained unchanged for up to 48 h although a slight decrease in
Table 1 Phagocytosis and subsequent phagosomelysosome fusion in murine macrophages infected with Tn5 mutant of S. typhimurium Strain 5170 (wild-type) 5635 (5170 : :Tn5)
Phagocytic index
Fusion index
218 .6±29 .0 247 .2±20 .4
4 .51+0 .50 54 .63±9 .06
Sulphorhodamine-labelled macrophages (1 x 10 5 cells/well) were infected with bacteria at a ratio of 1 :5 in the presence of 10% normal fresh serum . After 1 h incubation, phagocytosis and phagosomelysosome fusion were assayed . The values represent the means±SE of four experiments .
Pathogenesis of S. typhimurium
31 9
A
B
Fig . 1 . Fluorescence micrographs of sulphorhodamine-stained macrophages . (a) Macrophage prelabelled with sulphorhodamine were incubated with S . typhimurium 5170 at 37 ° C for 60 min . Macrophages contained many sulphorhodamine-stained lysosomes surrounding dark spaces (arrowhead) which were unstained bacteria in non-fused phagosomes (X900) . (b) Macrophages were infected with S . typhimurium 5635 in the same manner . A number of bacteria were stained with sulphorhodamine (arrow), indicating the diffusion of the lysosome marker dye into phagosomes (x 900) .
or 5635 (0) at a
32 0
Y . Ishibashi et al.
4
• •
3
2 •
1
48 24 Time after infection (h) Fig . 2 . Intracellular killing of Tn5 mutant of S . typhimurium by cultured murine macrophages in vitro . Macrophage monolayers (5x105 cells/well) were infected with S . typhimurium 5170 ( •) ratio of 1 :1 in the presence of 10% fresh serum . After 1 h incubation, infected macrophages were washed with RPM 11640 containing 5 pg/ml of gentamicin to remove the extracellular bacteria and further incubated at 37°C in 5% C02 . At various time intervals, the number of viable bacteria in macrophages were determined by colony counting after cell lysis with PBS containing 0 .05% triton x-100 . The values represent the mean±SE of five experiments . 1
6
the first 6 h was observed . In contrast, the viable number of the mutant strain within macrophages continued to decline over the entire 48 h infection period . We also examined the virulence of the mutant in mice in vivo . Mice were infected intraperitoneally (i .p .) or orally with a serial dilution of S . typhimurium 5635, and then 50% lethal dose (LD50) values were determined after 3 weeks . As shown in Table 2, LD50 values of the mutant strain were almost similar to those of parental strain . Mice infected by the same route displayed similar average times to death regardless of the challenge strain . No remarkable difference in the symptom was observed between parent strain and the mutant-infected mice . The possibility that the mutant strain reverted to wild-type in vivo was examined in parallel with the LD50 determinations . Additional three mice were challenged i .p . with 102 cfu of 5635 strain and then killed at 5 days after challenge . Their spleens were homogenized, appropriately diluted with saline and plated on L-agar with or without
Table 2 Virulence of Tn5 mutant of S . typhimurium to mice in vivo i .p .
P.O .
Strain
LD50
Average time a to death (day)
LD50
Average time to death (day)
5170 (wild-type) 5635 (5170 : :Tn5)
70 30
7 8
1 .2 x 105 9 .0x10°
11 11
Mice were infected intraperitoneally (i .p .) or orally (P .O .) with a serial dilution of S. typhimurium, and then LD50 values were determined after 3 weeks . The values represent the means of two experiments . Data taken 3 weeks post-challenge for mice receiving approximately 1 LD50 .
Pathogenesis of S. typhimurium
321
kanamycin (25,ug/ml) . Viable counts were found to be almost the same regardless of the presence of kanamycin, indicating that the mutant strain 5635 is still carrying the transposon insertion .
Discussion Our previous report has shown that S. typhimurium is capable of inhibiting phagosomelysosome fusion in murine macrophages after ingestion, suggesting that the fusion inhibitory function may be a virulence factor of S . typhimurium . To find an answer, we now screened a mutant lacking the fusion inhibitory function from a bank of Tn5 insertion mutants of S. typhimurium 5170 . Among 2500 mutants, only one mutant was found to be defective in the fusion inhibitory function . The mutant 5635 had no change in LPS composition, mobility and auxotrophic requirement . It has been reported that the virulence-associated plasmids in Salmonellae encode several virulence factors ."-" In our study, the transposon insertion appeared not to be in the plasmid but in the chromosome as determined by Southern hybridization using Tn5fragment as a probe (data not shown) . Horwitz" has previously demonstrated that the mutant strains of Legione//a pneumophila lacking phagosome-lysosome fusion inhibitory function are able to survive but not grow within phagocytes, and that these mutant strains are avirulent in vivo . Our study found that mutant 5635 was significantly sensitive to the intracellular killing by cultured macrophages as compared with the parental strain . However, the loss of the fusion inhibitory function was not associated with reduction of virulence in mice in vivo . These findings demonstrated that the fusion inhibitory function might not play a critical role for the net virulence of S . typhimurium, although it might contribute to at least a part of the intracellular survival within macrophages . Virulence of S. typhimurium has been attributed to various factors, 18 including resistance to reactive oxygen metabolites 19 and resistance to bactericidal peptides such as defensin . 20 Although the relative contribution of these mechanisms to virulence of S . typhimurium is far from being fully understood, it seemed likely that total pathogenesis of S . typhimurium might result from the complex of multiple virulence factors rather than a single virulence factor. An alternative interpretation might be possible . Lin et a/. 4 have demonstrated that the virulent strain of S . typhimurium is easily killed by phagocytes, but can efficiently grow in extracellular locations . It therefore seemed likely that an efficient extracellular growth of S . typhimurium in vivo might overcome the intracellular killing by macrophages even if the mutant strain had a defect in the fusion inhibitory function . Further analysis of the gene and its product(s) with regard to the fusion inhibitory function may provide an important information for understanding the interaction between S . typhimurium and host macrophages at the molecular level .
Materials and methods
Animals. Male BALB/c mice, aged 6 weeks, were used . Bacteria . Salmonella typhimurium 5170 wild-type, an isolate from a field case of calf enteritis, was kindly provided by Dr N . Terakado of the National Institute of Animal Health . It was grown in Luria broth (LB) at 37°C and harvested during exponential growth . Bacterial concentrations were determined by measuring the turbidity at 660 nm' and adjusted to desired concentration in Dulbecco's phosphate buffered saline (PBS) . Isolation of Tn5 mutants . Tn5 insertions were made by mating a temperature-sensitive
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R388ts : :Tn5 into 5170 at 32 ° C and selecting trimetprim-sensitive and kanamycin-resistant derivatives on Luria plates at 42°C as described previously . 21 Isolated mutants were cultured with LB or Luria agar plates in the presence of kanamycin (25 pg/ml) to prevent the accumulation of revertants . Reagents and media . RPM11640 medium and Dulbecco's PBS were obtained from Nissui Seiyaku Co ., Ltd, Tokyo, Japan . Gentamicin was purchased from Dainippon Seiyaku Co ., Ltd, Osaka, Japan . Sulphorhodamine 101 was purchased from Sigma Chemical Co ., St Louis, MO, U .S .A . Preparation of macrophages . Resident peritoneal macrophages were obtained by washing the peritoneal cavity with RPMI1640 containing 5 pg/ml of gentamicin (GM) and 5 U/ml of heparin as described previously .' To obtain macrophage monolayers, 100 PI of the macrophage suspensions (1 x106 cells/ml) were plated on eight-well HTC slides (Cel-Line Inc ., Newfield, NJ) and incubated for 1 h at 37°C . After incubation, macrophage monolayers (1 x 105 cells/well) were rinsed and cultured with RPMI1640-GM containing 10% foetal bovine serum (FBS) for 2 days at 37°C in 5% CO 2 before use . For intracellular killing assay, the macrophage monolayers (5 x 10 5 cells/well) were prepared in 24-well culture dishes in the same way . Assay for phagosome-lysosome fusion and phagocytosis . Phagosome-lysosome fusion in macrophages was assessed by using fluorescent vital dyes as described previously . 5.22 In brief, sulphorhodamine was used as a fluorescent marker for monitoring phagosome-lysosome fusion .' Macrophage monolayers were labelled with 200 Mg/ml of sulphorhodamine in RPMI1640 overnight at 37°C, and then rinsed with RPM11640 . Macrophages were infected with bacteria (macrophage to bacteria ratio of 1 :5) in a total volume of 100 pl of RMPI1640 containing 10% normal fresh serum . After incubation, macrophages were washed three times with RPM 11640GM and allowed to air dry, followed by UV-irradiation for 5 min to kill the bacteria . The activity of phagosome-lysosome fusion was determined by observing sulphorhodamine-stained bacteria under fluorescence microscope . Subsequently, macrophage monolayers were fixed with 4% paraformaldehyde, stained with Giemsa, and then phagocytic activity was determined by observing intracellular bacteria by light microscope . The fusion index and the phagocytic index were defined as the percentage positive fusion multiplied by the average number of fused phagosome per cell and the percentage positive phagocytosis multiplied by the average number of phagocytized bacteria per cell, respectively .' Intracellular killing by macrophages . Intracellular killing of bacteria by murine macrophages was assessed according to the method of Lissner 23 with a slight modification . The minimum inhibitory concentrations (MICs) of gentamicin for S . typhimurium strains were determined in RPMI1640 medium containing 10% FBS as described previously ." The MIC of gentamicin for both S. typhimurium 5170 and Tn5 insertion mutant was 2 .5 pg/ml . Thus, the concentration of 5 .0 pg/ml of gentamicin was used for the prevention of extracellular growth of bacteria . Macrophage monolayers (5x 10 5 cells/well) were infected with bacteria at a ratio of 1 :1 in the presence of 10% fresh serum . After 1 h incubation, infected macrophages were washed with RPM 11640 containing 5 pg/ml of gentamicin to remove the extracellular bacteria, and further incubated at 37°C in 5% CO 2 . After incubation, the number of viable bacteria in macrophages were determined by colony counting after cell lysis by PBS containing 0 .05% triton x-1 00 . Determination of LD 50 values. Animal inoculations for the determination of the LD 50 values were performed as described previously ." Five mice were housed in a cage . For intraperitoneal inoculation, mice were infected i .p . with 0 .2 ml of bacterial suspension serially diluted in buffered saline containing 0 .1% gelatin, and then LD 50 values were determined after 3 weeks . For oral challenge, mice were starved for food and water for 6 h and then fed 50 pl of 10% sodium bicarbonate, followed by 20 pl of bacterial suspension . LD 50 titers were calculated by the method of Reed and Muench 21 from results obtained from five mice per inoculum dose .
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