| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 VA Greater Los Angeles Healthcare System, Research Service, Los Angeles, CA 90073, USA
2 UCLA David Geffen School of Medicine, Los Angeles, CA 90095, USA
3 Biomanguinhos, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Rio de Janeiro, Brazil
4 Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, New York, NY 10021, USA
5 Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil
Correspondence
James Matsunaga
jamesm{at}ucla.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Present address: UC Davis School of Veterinary Medicine, Davis, CA 95616, USA.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Leptospira interrogans is one of 14 genomospecies of pathogenic Leptospira and is the cause of endemic disease and large outbreaks of leptospirosis worldwide (Brenner et al., 1999; Levett et al., 2006). Outbreaks involving L. interrogans have been associated with flooding in rat-infested urban slums (Ko et al., 1999). L. interrogans resides chronically in the lumen of rat kidney tubules and is released within urine into the environment, where it can survive for weeks (Crawford et al., 1971; Gordon Smith & Turner, 1961; Ryu & Liu, 1966). Humans exposed to Leptospira in the environment can be infected through breaks in skin or mucous membranes. After breaching the epithelium or mucosa, leptospires migrate to the bloodstream and disseminate to numerous organs. Flu-like symptoms including fever and myalgia characterize early acute-phase infection. Severe symptoms including jaundice, renal failure and pulmonary haemorrhage are subsequently observed in 5–10 % of cases (Bharti et al., 2003; McBride et al., 2005).
Survival of L. interrogans inside and outside of the host is likely to require the expression of different sets of gene products. Little is known about which environmental cues signal L. interrogans to modify its gene expression patterns during infection. At least four components of pathogenic Leptospira have been shown to be differentially expressed when leptospires are transferred from culture medium into a host animal: LigA, Sph2 (Lk73.5), LipL36 and lipopolysaccharide (LPS) O antigen. The extracellular matrix (ECM)-binding proteins LigA and LigB, the haemolysin Sph2, and LPS are potential virulence determinants (Artiushin et al., 2004; Matsunaga et al., 2003; Nally et al., 2005; Choy et al., 2007). Initial studies of LigA and Sph2 failed to detect either protein in L. interrogans lysates (Artiushin et al., 2004; Palaniappan et al., 2002), although later studies revealed low levels of both in lysates and culture supernatant fluids (Matsunaga et al., 2005; Zhang et al., 2005). However, infected humans and horses, respectively, produced antibody to these proteins, indicating that expression of these proteins occurs in the host. Additionally, LigA was detected in kidneys of hamsters infected with L. interrogans but not in cultured leptospires (Palaniappan et al., 2002). In contrast, LipL36, an abundant outer-membrane lipoprotein in in vitro-cultured leptospires, was not detected in leptospires residing in the kidney tubule of an experimentally infected hamster (Haake et al., 1998). Similarly, L. interrogans isolated from the liver of an acutely infected guinea pig did not contain detectable levels of O antigen, which is found on the outer surface of cultivated leptospires (Nally et al., 2005).
The lack of efficient tools for genetic manipulaton of pathogenic Leptospira and intrinsic difficulties in characterizing leptospiral gene expression during infection of experimental animals have impeded elucidation of the role of host-adapted leptospiral determinants in virulence. Furthermore, little progress has been made in identifying culture conditions that reproduce the changes in leptospiral gene expression observed during infection. The diminished LipL36 levels observed in vitro by temperature upshift and iron limitation may explain the inability to detect LipL36 in the kidneys of infected hamsters (Cullen et al., 2002; Haake et al., 1998; Nally et al., 2001b). Recent microarray and proteomic experiments have revealed changes in the levels of numerous transcripts and several outer-membrane proteins in response to different culture conditions (Cullen et al., 2002; Lo et al., 2006; Nally et al., 2001b; Qin et al., 2006). However, whether these genes behave similarly during infection of a mammalian host is unknown.
Leptospira outside of the host is typically found in environments such as fresh water or moist soil, where osmolarity is lower than that found within mammals (Kratz et al., 2004; Miller & Wood, 1996). We recently demonstrated that levels of cellular LigA and LigB and extracellular LigA are increased when the ionic strength of leptospiral culture medium is increased to achieve the osmolarity found in the host (Matsunaga et al., 2005). The change in osmolarity that occurs as L. interrogans enters the host from a freshwater environment may be a signal to the leptospires to increase expression of ligA, ligB, and other putative virulence genes and reduce expression of gene products necessary for survival outside of the host. In this study, we systematically examined expression of leptospiral genes known to be regulated by mammalian host signals. We found that osmolarity, irrespective of whether ionic or non-ionic solutes were used, influenced cellular levels of LigA, LigB, Sph2 and LipL36. In contrast, the amount of LigA and Sph2 detected in culture supernatant fluids was differentially affected by the method used to raise the osmolarity. Thus, osmolarity can affect expression of leptospiral genes at different steps in gene expression.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Nascimento et al., 2004a). Virulence was maintained by infection and reisolation of the strain from Golden Syrian hamsters. L. interrogans was maintained in EMJH medium supplemented with 1 % rabbit serum and 100 µg 5-fluorouracil ml–1 at 30 °C (Ellis & Thiermann, 1986). Cell density was determined by direct counts of motile bacteria by darkfield microscopy. Unless indicated otherwise, all experiments were performed with L. interrogans grown to 2–8x108 ml–1.
Plasmid DNA.
The portion of the sph2 gene encoding codons 27 through 190 was cloned into an expression vector as follows. The forward primer 5'-CACCGAAAAAGAATCCTCATATAAGGATTTATTTACTTCG-3' and the reverse primer 5'-TCATACGTAATCTGATTTTGAAATTCGTTTTGC-3' were used to amplify the sph2 gene fragment from L. interrogans genomic DNA by PCR with Pfu DNA polymerase (Finnzymes). The PCR product was inserted directionally into pET151/D-TOPO (Invitrogen) to generate the plasmid pTOPO-Sph2(27-190). Similarly, the portion of sph2 corresponding to codon 27 through the stop codon was cloned into pET151/D-TOPO to generate pTOPO-Sph2(27-623). The sequence of the reverse primer used with the forward primer described above was 5'-TTAGCGATAAATAAGATCCGCACTCCA-3'.
Antisera.
Lig, LipL36, and LipL41 rabbit antisera have been described (Haake et al., 1998; Matsunaga et al., 2003; Shang et al., 1996). Sph2 rabbit antiserum was raised as follows. Plasmid pTOPO-Sph2(27-190) was transformed into Escherichia coli BLR(DE3)/pLysS (EMD Biosciences), and expression of the His6-Sph2(27-190) protein was induced with 0.5 mM IPTG. Purification of the recombinant protein and immunization of New Zealand rabbits were performed as previously described (Matsunaga et al., 2005). The immunization protocol was approved by the VA Greater Los Angeles Institutional Animal Care and Use Committee.
Immunoblot analysis.
Culture supernatant fluid was collected following centrifugation of 5x108–1x109 leptospires for 4 min at 9000 g in a Beckman Coulter Microfuge 18 centrifuge. Leptospiral proteins were isolated from the culture supernatant by immunoprecipitation with specific rabbit antisera, as described previously (Matsunaga et al., 2005). Cell pellets and immunoprecipitated protein were subjected to immunoblot analysis as described (Matsunaga et al., 2005). Lig, Sph2, LipL41 and LipL36 antisera were used at titres of 1 : 2000, 1 : 1000, 1 : 10 000 and 1 : 1000, respectively.
RNA extraction.
L. interrogans cultures were chilled in a dry ice-ethanol bath and centrifuged at 10 000 r.p.m. for 15 min in a Sorvall SS34 rotor. RNA was extracted from the bacteria with TRIzol reagent (Invitrogen) and treated with 2 U Turbo-DNase (Ambion) in a final volume of 100 µl for 30 min at 37 °C, as directed by the manufacturer. An additional 2 U Turbo-DNase was then added, and incubation was continued for another 30 min. DNase was removed by phenol/chloroform extraction followed by ethanol precipitation.
RT-PCR.
Two micrograms of leptospiral RNA was hybridized to random nonamer primers (Sigma) and cDNA was synthesized with Omniscript reverse transcriptase, as specified by the manufacturer (Qiagen). The cDNA was amplified with Taq DNA polymerase (Qiagen) with the gene-specific primer pairs shown in Table 1. Primers were designed with Primer Premier 5 (Premier Biosoft International). PCR reactions were examined by electrophoresis in 1.5 % agarose gels. The 1 kb DNA ladder was obtained from Gene Choice and Promega.
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Western blot analysis of L. interrogans grown overnight in EMJH supplemented with 120 mM sodium chloride demonstrated an increase in LigA and LigB (Fig. 1, lane 1 vs 3) above levels observed in L. interrogans grown in EMJH, as demonstrated previously (Matsunaga et al., 2005). Similarly, when L. interrogans was incubated in EMJH with 240 mM glucose or sucrose to attain physiological osmolarity, LigA and LigB levels increased (Fig. 1, lanes 5 and 7), demonstrating for the first time that Lig protein levels respond to the osmolarity and not the ionic strength of the environment.
|
, lane 4) but not to glucose or sucrose (Fig. 1, lanes 6 and 8). LigA was immunoprecipitated with Lig antiserum prior to Western blot analysis to avoid distortion of the gel caused by the high concentration of albumin in EMJH. A control experiment was performed to rule out the possibility that glucose and sucrose inhibited immunoprecipitation of LigA. L. interrogans was incubated overnight in EMJH with 120 mM sodium chloride to provide a source of LigA. Culture supernatants were dialysed against EMJH and supplemented with sucrose or glucose at a final concentration of 240 mM. Sucrose and glucose had no effect on the ability to immunoprecipitate LigA (data not shown), indicating that the reduction of extracellular LigA signal was not due to the inability to immunoprecipitate the protein in the presence of non-ionic solutes. LipL41 levels were not affected by osmolarity, as demonstrated previously (Matsunaga et al., 2005).
Another leptospiral gene induced by host signals is sph2, which encodes a sphingomyelinase (Artiushin et al., 2004; Zhang et al., 2005). To determine whether sph2 could be induced by osmolarity, RT-PCR was performed to examine sph2 transcript levels in L. interrogans grown overnight in EMJH and in EMJH supplemented with 100 mM sodium chloride or 200 mM sucrose. The sph2 mRNA was not detected in L. interrogans grown in EMJH (Fig. 2, lane 7). However, sph2 mRNA was detected in L. interrogans grown in salt- or sucrose-supplemented EMJH (Fig. 2, lanes 8 and 9). Transcripts for ligA and ligB also increased when EMJH was supplemented with sodium chloride or sucrose (Fig. 2, lanes 1 vs 2 and 3, 4 vs 5 and 6). The control lipL41 transcript was detected under all growth conditions (Fig. 2, lanes 10–12). When reverse transcriptase was omitted from the reaction, PCR products were not detected, demonstrating adequate digestion of genomic DNA (data not shown).
|
). The antiserum strongly recognized full-length recombinant Sph2 protein, which has an apparent molecular mass of 89 kDa, larger than the calculated molecular mass of 71.7 kDa (Fig. 3a, lane 4). However, immunoblots of L. interrogans grown overnight in EMJH probed with Sph2 antiserum revealed a much smaller species of 63 kDa (Fig. 3a, lane 1). On the other hand, when the bacteria were incubated in EMJH supplemented with sodium chloride or sucrose, a band that co-migrated with the recombinant Sph2 protein appeared (Fig. 3a, lanes 2 and 3). The 63 kDa band is likely to be one of the other sphingomyelinase-like proteins expressed by L. interrogans (Zhang et al., 2005). There was no effect on LipL41 levels, as expected (Fig. 3b, lanes 1–3).
|
, lane 1). These bands appear to represent a processed version of the 89 kDa cell-associated Sph2 since the 63 kDa band detected in cell lysates was too small to be a precursor of the two bands. The level of expression of these species was significantly increased in supernatants from leptospires treated with sodium chloride (Fig. 3c, lane 2). Culture supernatant from L. interrogans grown in EMJH with sucrose was also examined for induction of extracellular Sph2. In contrast to the minimal change observed with LigA (Fig. 3d, lane 1 vs 3), sucrose treatment induced the release of extracellular Sph2 at similar levels to that observed in supernatants from salt-treated leptospires (Fig. 3c, lane 3). LipL41 was not detected in the culture supernatant, demonstrating that harvesting the bacteria was not accompanied by cell lysis (Fig. 3d, lanes 4–6).
The kinetics of induction of Lig and Sph2 protein expression by sodium chloride was examined. A culture of L. interrogans growing in EMJH was transferred to EMJH supplemented with 100 mM sodium chloride at time zero. Cells and culture supernatant were collected 1, 2, 4, 6 and 23 h after osmotic upshift. An increase in LigA, LigB and Sph2 was detected 1 h following osmotic upshift (Fig. 4a and b, lane 1 vs 2). Maximum levels of LigA and LigB were reached after 6 h (Fig. 4a, lane 5). Sph2 levels also increased over 6 h but diminished by 23 h (Fig. 4b, lane 6). There was no effect of sodium chloride on LipL41 levels or the 63 kDa band that cross-reacted with the Sph2 antiserum. Culture supernatant fluid collected at each time point was also examined for LigA and Sph2 signal by immunoprecipitation and subsequent Western blot analysis. Both signals exhibited a gradual increase over several hours following osmotic upshift. A separate experiment demonstrated that Sph2 levels were regulated by growth phase in EMJH with 80 mM sodium chloride (Fig. 4c), which may explain the diminished levels of Sph2 at 23 h in Fig. 4(b).
|
). Five L. interrogans cultures having different starting cell densities were initiated in EMJH containing 80 mM sodium chloride and allowed to reach a density of 4–5x108 ml–1 following 4, 24, 48, 96 and 144 h of incubation. Under these conditions, a gradual decrease in LipL36 levels was observed, whereas LipL41 levels were unaffected (Fig. 5a). In a separate experiment, when L. interrogans was allowed to double twice in EMJH containing 100 mM sodium chloride or 200 mM sucrose, LipL36 levels dropped compared to that in leptospires incubated in EMJH alone (Fig. 5b, lane 1 vs 2 and 3). Levels of the abundant outer-membrane proteins LipL21 (Cullen et al., 2003), LipL32 (Haake et al., 2000) and LipL46 (Matsunaga et al., 2006) were not affected by the increase in osmolarity (data not shown).
|
, lanes 1–3). As expected, no effect of osmolarity on lipL41 transcript levels was detected (Fig. 6, lanes 4–6).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Haake et al., 1998; Matsunaga et al., 2003; Palaniappan et al., 2002), suggesting that L. interrogans senses osmolarity to regulate expression of ligA, ligB, sph2 and lipL36 during transition from a freshwater environment into a host. Both sodium chloride and sucrose regulate expression of these genes, indicating that the true signal sensed by L. interrogans is osmolarity, not ionic strength. Such a mode of gene regulation is ideal for a pathogen whose life cycle involves environments with considerable osmotic differences. To our knowledge, osmotically regulated genes have not been identified in other spirochaetes. We have recently used whole-genome microarrays to identify additional leptospiral genes induced by an upshift to physiological osmolarity (Matsunaga et al., 2007).
In our time-course study, we demonstrated the accumulation of the Lig and Sph2 proteins within hours following osmotic upshift (Fig. 4). Rapid accumulation of Lig proteins is consistent with the detection of IgM antibody against Lig during the acute stage of leptospirosis in 92 % of patients (Croda et al., 2007). Previous studies have examined the effects of environmental changes on leptospiral gene expression at longer time points following shift in culture conditions (Cullen et al., 2002; Lo et al., 2006; Matsunaga et al., 2005; Nally et al., 2001a, b; Qin et al., 2006). Our results suggest that rapid accumulation of the putative virulence determinants LigA, LigB and Sph2 may be involved in the initial stages of host infection.
Similar to the results of Artiushin et al., (2004), we were unable to detect Sph2 by immunoblot analysis of L. interrogans grown in EMJH (Fig. 3a). The requirement for growth of L. interrogans at physiological osmolarity for sph2 induction is a likely explanation of the inability of Artiushin et al. to detect the sphingomyelinase. In contrast, Zhang et al. (2005) detected Sph2 in L. interrogans lysates. It is possible that the 10 % rabbit serum in the Korthof medium used by Zhang et al. (2005) to generate some of their data increased the osmolarity of the medium sufficiently to raise Sph2 to detectable levels. The cross-reacting 63 kDa band (Fig. 3a) could be SphH, which retains sequence similarity with the Sph2 fragment used to generate the antiserum. In addition, microarray analysis showed that sphH transcripts exhibited by far the strongest signal among the sphingomyelinase-like genes in L. interrogans grown in EMJH, again consistent with SphH being the 63 kDa species (Matsunaga et al., 2007). Despite its extensive sequence similarity to Sph2, SphH lacks sphingomyelinase activity and instead forms pores in the mammalian cytoplasmic membrane (Lee et al., 2000, 2002).
Earlier studies revealed leptospiral proteins in the culture supernatant, including LigA, sphingomyelinase and Hap1 (LipL32) (Branger et al., 2005; Matsunaga et al., 2005; Zhang et al., 2005). Several of these extracellular proteins elicited a humoral immune response during infection (Zuerner et al., 1991). The osmotically regulated 68 and 76 kDa bands detected in culture supernatant fluids with Sph2 antiserum (Fig. 3) are too large to be derived from the constitutively expressed 63 kDa cell-associated band that cross-reacted with the antiserum. Further, RT-PCR (data not shown) and microarray analysis demonstrated that among the five genes encoding sphingomyelinase-like proteins, only transcript from sph2 was regulated by salt (Matsunaga et al., 2007). Thus, the 68 and 76 kDa bands most likely derive from Sph2.
Among the five sphingomyelinase-like genes, only sph2 transcript levels are affected by salt (Matsunaga et al., 2007), suggesting that the genes are regulated by different environmental signals. The different sphingomyelinases may perform distinct functions and exhibit tissue-specific expression during infection. For example, the osmotic induction of sph2 may have a role in protecting L. interrogans from osmotic upshifts, since choline, a potential byproduct of phosphocholine produced by sphingomyelinase activity, is converted by many bacteria into the osmoprotectant glycine betaine (Shortridge et al., 1992). Although a gene encoding the choline transporter has not been found in the L. interrogans genome, genes encoding homologues of enzymes that convert choline to glycine betaine are present (Nascimento et al., 2004a; Ren et al., 2003).
L. interrogans harbours numerous genes encoding homologues of signal transduction proteins, including 48 sensors and 38 response regulators that are members of two-component regulatory systems, 12 membrane-bound adenylate cyclases, and 11 extracytoplasmic sigma factors (Galperin, 2005, 2006; Nascimento et al., 2004b). As shown in other bacteria, some of these signalling proteins may be involved in transducing signals to the genetic regulatory apparatus following osmotic upshift (Heusipp et al., 2003; Jubelin et al., 2005; Kimura et al., 2002, 2005). Other than the kdpDE genes encoding the two-component regulatory system that regulates expression of a high-affinity potassium transporter, L. interrogans lacks obvious homologues of osmotically controlled signal transduction proteins found in other bacteria (Nascimento et al., 2004a; Ren et al., 2003).
The post-transcriptional effect of osmolarity is a novel finding among spirochaetes. Enhanced LigA release occurred when the osmolarity was increased with sodium chloride but not with non-ionic solutes (Fig. 1). Because most of the osmolarity of tissue fluids is accounted for by sodium chloride, the results observed with sodium chloride are more relevant to host infection. Release of LigA at physiological osmolarity was not due to general proteolytic release of surface proteins, as LigB and LipL41 were not released. We therefore hypothesize that large amounts of LigA are released during infection. It is possible that ionic strength affects the conformation of LigA, a putative LigA protease, or both, permitting cleavage when the salt concentration is high. The failure to observe an increase of LigA levels following addition of non-ionic solutes raises the possibility that LigA release is regulated by unknown factors in the variety of niches found in the host. For example, protease inhibitors, which are abundant in human plasma (Travis & Salvesen, 1983), may affect LigA release in vivo. In contrast to the results observed for LigA, Sph2 release occurred whether the osmolarity was increased with sodium chloride or sucrose. These results suggest that the mechanisms of LigA and Sph2 release into the environment differ, although release of both involves proteolytic cleavage. Possible roles of adhesin release include enhancing dissemination of bacteria and control of immune cell activity (Abramson et al., 2001; Coutte et al., 2003; McGuirk & Mills, 2000).
LipL36 is subject to multiple environmental controls, including temperature, iron availability and growth phase (Cullen et al., 2002; Haake et al., 1998; Nally et al., 2001b). An increase in incubation temperature caused a reduction in LipL36 levels (Nally et al., 2001b) without affecting lipL36 transcript levels (Lo et al., 2006). In contrast, osmotic upshift caused diminished lipL36 expression at both the RNA and protein levels. Hence, regulation of lipL36 expression appears to be molecularly complex.
In conclusion, we have demonstrated that raising the osmolarity of leptospiral growth medium to near-physiological levels reproduced the changes in levels of Lig, Sph2 and LipL36 that are observed when L. interrogans infects a host mammal. This observation supports the notion that the ability to sense external osmolarity is important in correctly adjusting gene expression levels during the early stages of infection. The rapid increase in the levels of the putative virulence determinants LigA, LigB and Sph2 observed following upshift to physiological osmolarity in vitro may reflect events that occur at the early stages of L. interrogans infection of a susceptible host. Finally, osmolarity can mediate its effects via post-transcriptional mechanisms, as observed for extracellular LigA.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: L. S. Frost
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Artiushin, S., Timoney, J. F., Nally, J. & Verma, A. (2004). Host-inducible immunogenic sphingomyelinase-like protein, Lk73.5, of Leptospira interrogans. Infect Immun 72, 742–749.
Bharti, A. R., Nally, J. E., Ricaldi, J. N., Matthias, M. A., Diaz, M. M., Lovett, M. A., Levett, P. N., Gilman, R. H., Willig, M. R. & other authors (2003). Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3, 757–771.[CrossRef][Medline]
Branger, C., Chatrenet, B., Gauvrit, A., Aviat, F., Aubert, A., Bach, J. M. & Andre-Fontaine, G. (2005). Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1. Infect Immun 73, 4062–4069.
Brenner, D. J., Kaufmann, A. F., Sulzer, K. R., Steigerwalt, A. G., Rogers, F. C. & Weyant, R. S. (1999). Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. Int J Syst Bacteriol 49, 839–858.
Choy, H. A., Kelley, M. M., Chen, T. L., Moller, A. K., Matsunaga, J. & Haake, D. A. (2007). Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. Infect Immun 75, 2441–2450.
Coutte, L., Alonso, S., Reveneau, N., Willery, E., Quatannens, B., Locht, C. & Jacob-Dubuisson, F. (2003). Role of adhesin release for mucosal colonization by a bacterial pathogen. J Exp Med 197, 735–742.
Crawford, R. P., Heinemann, J. M., McCulloch, W. F. & Diesch, S. L. (1971). Human infections associated with waterborne leptospires, and survival studies on serotype pomona. J Am Vet Med Assoc 159, 1477–1484.[Medline]
Croda, J., Ramos, J. G., Matsunaga, J., Queiroz, A., Homma, A., Riley, L. W., Haake, D. A., Reis, M. G. & Ko, A. I. (2007). Leptospira immunoglobulin-like proteins as a serodiagnostic marker for acute leptospirosis. J Clin Microbiol 45, 1528–1534.
Cullen, P. A., Cordwell, S. J., Bulach, D. M., Haake, D. A. & Adler, B. (2002). Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect Immun 70, 2311–2318.
Cullen, P. A., Haake, D. A., Bulach, D. M., Zuerner, R. L. & Adler, B. (2003). LipL21 is a novel surface-exposed lipoprotein of pathogenic Leptospira species. Infect Immun 71, 2414–2421.
Ellis, W. A. & Thiermann, A. B. (1986). Isolation of Leptospira interrogans serovar bratislava from sows in Iowa. Am J Vet Res 47, 1458–1460.[Medline]
Galperin, M. Y. (2005). A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts. BMC Microbiol 5, 35[CrossRef][Medline]
Galperin, M. Y. (2006). Structural classification of bacterial response regulators: diversity of output domains and domain combinations. J Bacteriol 188, 4169–4182.
Gordon Smith, C. E. & Turner, L. H. (1961). The effect of pH on the survival of leptospires in water. Bull Wld Hlth Org 24, 35–43.
Haake, D. A., Martinich, C., Summers, T. A., Shang, E. S., Pruetz, J. D., McCoy, A. M., Mazel, M. K. & Bolin, C. A. (1998). Characterization of leptospiral outer membrane lipoprotein LipL36: downregulation associated with late-log-phase growth and mammalian infection. Infect Immun 66, 1579–1587.
Haake, D. A., Chao, G., Zuerner, R. L., Barnett, J. K., Barnett, D., Mazel, M., Matsunaga, J., Levett, P. N. & Bolin, C. A. (2000). The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect Immun 68, 2276–2285.
Heusipp, G., Schmidt, M. A. & Miller, V. L. (2003). Identification of rpoE and nadB as host responsive elements of Yersinia enterocolitica. FEMS Microbiol Lett 226, 291–298.[CrossRef][Medline]
Jubelin, G., Vianney, A., Beloin, C., Ghigo, J. M., Lazzaroni, J. C., Lejeune, P. & Dorel, C. (2005). CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli. J Bacteriol 187, 2038–2049.
Kimura, Y., Mishima, Y., Nakano, H. & Takegawa, K. (2002). An adenylyl cyclase, CyaA, of Myxococcus xanthus functions in signal transduction during osmotic stress. J Bacteriol 184, 3578–3585.
Kimura, Y., Ohtani, M. & Takegawa, K. (2005). An adenylyl cyclase, CyaB, acts as an osmosensor in Myxococcus xanthus. J Bacteriol 187, 3593–3598.
Ko, A. I., Reis, M. G., Dourado, C. M., Johnson, W. D. & Riley, L. W. (1999). Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 354, 820–825.[Medline]
Kratz, A., Ferraro, M., Sluss, P. M. & Lewandrowski, K. B. (2004). Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Laboratory reference values. N Engl J Med 351, 1548–1563.
Lee, S. H., Kim, K. A., Park, Y. G., Seong, I. W., Kim, M. J. & Lee, Y. J. (2000). Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai. Gene 254, 19–28.[CrossRef][Medline]
Lee, S. H., Kim, S., Park, S. C. & Kim, M. J. (2002). Cytotoxic activities of Leptospira interrogans hemolysin SphH as a pore-forming protein on mammalian cells. Infect Immun 70, 315–322.
Levett, P. N., Morey, R. E., Galloway, R. L. & Steigerwalt, A. G. (2006). Leptospira broomii sp. nov., isolated from humans with leptospirosis. Int J Syst Evol Microbiol 56, 671–673.
Lo, M., Bulach, D. M., Powell, D. R., Haake, D. A., Matsunaga, J., Paustian, M. L., Zuerner, R. L. & Adler, B. (2006). Effects of temperature on gene expression patterns in Leptospira interrogans serovar Lai as assessed by whole-genome microarrays. Infect Immun 74, 5848–5859.
Matsunaga, J., Barocchi, M. A., Croda, J., Young, T. A., Sanchez, Y., Siqueira, I., Bolin, C. A., Reis, M. G., Riley, L. W. & other authors (2003). Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol Microbiol 49, 929–945.[CrossRef][Medline]
Matsunaga, J., Sanchez, Y., Xu, X. & Haake, D. A. (2005). Osmolarity, a key environmental signal controlling expression of leptospiral proteins LigA and LigB and the extracellular release of LigA. Infect Immun 73, 70–78.
Matsunaga, J., Werneid, K., Zuerner, R. L., Frank, A. & Haake, D. A. (2006). LipL46 is a novel surface-exposed lipoprotein expressed during leptospiral dissemination in the mammalian host. Microbiology 152, 3777–3786.
Matsunaga, J., Lo, M., Bulach, D. M., Zuerner, R. L., Adler, B. & Haake, D. A. (2007). Response of Leptospira interrogans to physiologic osmolarity: relevance in signaling the environment-to-host transition. Infect Immun 75, 2864–2874.
McBride, A. J., Athanazio, D. A., Reis, M. G. & Ko, A. I. (2005). Leptospirosis. Curr Opin Infect Dis 18, 376–386.[Medline]
McGuirk, P. & Mills, K. H. (2000). Direct anti-inflammatory effect of a bacterial virulence factor: IL-10-dependent suppression of IL-12 production by filamentous hemagglutinin from Bordetella pertussis. Eur J Immunol 30, 415–422.[CrossRef][Medline]
Miller, K. J. & Wood, J. M. (1996). Osmoadaptation by rhizosphere bacteria. Annu Rev Microbiol 50, 101–136.[CrossRef][Medline]
Nally, J. E., Artiushin, S. & Timoney, J. F. (2001a). Molecular characterization of thermoinduced immunogenic proteins Qlp42 and Hsp15 of Leptospira interrogans. Infect Immun 69, 7616–7624.
Nally, J. E., Timoney, J. F. & Stevenson, B. (2001b). Temperature-regulated protein synthesis by Leptospira interrogans. Infect Immun 69, 400–404.
Nally, J. E., Chow, E., Fishbein, M. C., Blanco, D. R. & Lovett, M. A. (2005). Changes in lipopolysaccharide O antigen distinguish acute versus chronic Leptospira interrogans infections. Infect Immun 73, 3251–3260.
Nascimento, A. L., Ko, A. I., Martins, E. A., Monteiro-Vitorello, C. B., Ho, P. L., Haake, D. A., Verjovski-Almeida, S., Hartskeerl, R. A., Marques, M. V. & other authors (2004a). Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J Bacteriol 186, 2164–2172.
Nascimento, A. L., Verjovski-Almeida, S., Van Sluys, M. A., Monteiro-Vitorello, C. B., Camargo, L. E., Digiampietri, L. A., Harstkeerl, R. A., Ho, P. L., Marques, M. V. & other authors (2004b). Genome features of Leptospira interrogans serovar Copenhageni. Braz J Med Biol Res 37, 459–477.[Medline]
Palaniappan, R. U., Chang, Y. F., Jusuf, S. S., Artiushin, S., Timoney, J. F., McDonough, S. P., Barr, S. C., Divers, T. J., Simpson, K. W. & other authors (2002). Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans. Infect Immun 70, 5924–5930.
Qin, J. H., Sheng, Y. Y., Zhang, Z. M., Shi, Y. Z., He, P., Hu, B. Y., Yang, Y., Liu, S. G., Zhao, G. P. & Guo, X. K. (2006). Genome-wide transcriptional analysis of temperature shift in L. interrogans serovar lai strain 56601. BMC Microbiol 6, 51[CrossRef][Medline]
Ren, S. X., Fu, G., Jiang, X. G., Zeng, R., Miao, Y. G., Xu, H., Zhang, Y. X., Xiong, H., Lu, G. & other authors (2003). Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature 422, 888–893.[CrossRef][Medline]
Ryu, E. & Liu, C. K. (1966). The viability of leptospires in the summer paddy water. Jpn J Microbiol 10, 51–57.[Medline]
Shang, E. S., Summers, T. A. & Haake, D. A. (1996). Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species. Infect Immun 64, 2322–2330.[Abstract]
Shortridge, V. D., Lazdunski, A. & Vasil, M. L. (1992). Osmoprotectants and phosphate regulate expression of phospholipase C in Pseudomonas aeruginosa. Mol Microbiol 6, 863–871.[CrossRef][Medline]
Travis, J. & Salvesen, G. S. (1983). Human plasma proteinase inhibitors. Annu Rev Biochem 52, 655–709.[CrossRef][Medline]
Zhang, Y. X., Geng, Y., Bi, B., He, J. Y., Wu, C. F., Guo, X. K. & Zhao, G. P. (2005). Identification and classification of all potential hemolysin encoding genes and their products from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai. Acta Pharmacol Sin 26, 453–461.[CrossRef][Medline]
Zuerner, R. L., Knudtson, W., Bolin, C. A. & Trueba, G. (1991). Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona. Microb Pathog 10, 311–322.[CrossRef][Medline]
Received 8 March 2007;
revised 30 May 2007;
accepted 6 June 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
