Environmental modulation of gene expression and pathogenesis in Yersinia.

The yersiniae are a useful model for understanding how environmental modulation of gene expression allows pathogens to inhabit a wide range of niches. This review follows the enteropathogenic yersiniae, Yersinia enterocolitica and Yersinia pseudotuberculosis, and the agent of plague, Yersinia pestis, through their life cycles, describing how adaptive gene expression may promote successful pathogenesis.

Use of UV-irradiated bacteriophage T6 to kill extracellular bacteria in tissue culture infectivity assays.

We have utilized 'lysis from without' mediated by UV-inactivated bacteriophage T6 to eliminate extracellular bacteria in experiments measuring the internalization, intracellular survival and replication of Yersinia pestis within mouse peritoneal macrophages and of Shigella flexneri within a human intestinal epithelial cell line. The technique we describe has the following characteristics: (a) bacterial killing is complete within 15 min at 37 degrees C, with a greater than 10(3)-fold reduction in colony-forming units (CFU); (b) bacteria within cultured mammalian cells are protected from killing by UV-inactivated T6; (c) the mammalian cells are not observably affected by exposure to UV-inactivated T6. This technique has several advantages over the use of antibiotics to eliminate extracellular bacteria and is potentially widely applicable in studies of the interactions between pathogenic bacteria and host phagocytic cells as well as other target tissues.

Interaction between Yersinia pestis YopM protein and human alpha-thrombin.

YopM, a 41.5 kDa virulence protein of Yersinia pestis is believed to have an anti-inflammatory role in bubonic plague. It has been shown previously that YopM binds human alpha-thrombin but not prothrombin and inhibits thrombin-induced platelet aggregation in vitro. In the present studies we carried out crosslinking reactions between purified YopM and alpha-thrombin or its blocked form FPR-alpha-thrombin in the presence of various competitors to identify where on thrombin YopM binds. We found that thrombin cleaves YopM at the C-terminus, indicating that this part of YopM must interact with thrombin's catalytic site. Hirudin, a 65 amino acid natural thrombin inhibitor, prevents both the YopM degradation and the formation of a ca. 75 kDa crosslinking complex between YopM and alpha-thrombin. A similar effect is observed when hirugen, a short peptide corresponding to hirudin's C-terminus (amino acids 58-64), is used as a synthetic thrombin inhibitor. A 15 bp long specific oligonucleotide known to block alpha-thrombin successfully competes with YopM for the thrombin-binding site, whereas a control, scrambled sequence aptamer does not. As these competitors block a fibrinogen binding site (also called anion binding exosite I), our crosslinking data indicate that YopM binds not only to the active site of alpha-thrombin but also to the abeI.

Unexpected results from the application of signature-tagged mutagenesis to identify Yersinia pestis genes required for adherence and invasion.

The signature-tagged mutagenesis (STM) method was applied in a protocol designed to identify genes required for Yersinia pestis invasion into epithelial cells. A library of 3060 mutants of Y. pestis CO99-3015 was made and assayed using an in vitro invasion assay with gentamicin protection. Initial results from the screen identified a set of 23 genes that might be required for invasion; however, screening of individual mutants for decreased invasion, even in a competition assay with the parent strain, failed to reveal obvious invasion defects. Altered colony character or size might have imposed a growth disadvantage for two of the mutants, which could possibly account for apparently decreased invasion. The sensitivity of the mutants to gentamicin was assayed to determine if the presence of the kanamycin-resistance cassette in the STM transposon changed the gentamicin resistance of the individual mutants. It was discovered that the mutants exhibited a variable range of resistance to killing by gentamicin, suggesting that the presence of the kanamycin-resistance cassette or the particular insertion mutation did in many cases affect the bactericidal potency of gentamicin. However, all mutants remained highly sensitive to growth inhibition in a disk assay on plates. These results may warrant precautions for use of kanamycin-resistance markers in studies with fully virulent Y. pestis, since gentamicin has been recommended for treatment of plague. Further, to use STM in the context of invasion assays, a selection other than gentamicin should be applied.

The low-Ca2+ response virulence regulon of human-pathogenic Yersiniae.

The Lcr is thought to function when the yersiniae infect their mammalian host (see Fig. 2). At present, all evidence points to roles of the Lcr in protecting the bacteria in extracellular niches. We can speculate that early in infection, the yersiniae experience a 37 degrees C extracellular, Ca(2+)-containing environment. Temperature-induction and down-modulation by Ca2+ would occur for Lcr genes, resulting in a cytotoxic and weakly anti-phagocytic surface on the bacteria. Yersiniae that are taken up into macrophages may find a low-Ca2+, inductive environment for expression and release of Yops and V antigen. Upon release from macrophages, the accumulated Yops may render the bacteria sufficiently anti-phagocytic to resist ingestion by neutrophils, and Lcr virulence proteins such as YopM, and perhaps V antigen and other Yops might act to dampen the inflammatory response and prevent secondary influxes of phagocytes. The abundant nucleotides from necrotic host cells undergoing nucleic acid breakdown might then promote extracellular growth. Even though Lcr virulence genes would be expressed at an intermediate level under these conditions, this may be sufficient for the continued undermining of the host natural defenses. It also is possible that there are additional environmental signals, not yet identified, that modulate Lcr virulence gene expression and permit strong expression in extracellular microenvironments that arise as a result of the host-yersiniae interactions.

The plasmid-encoded outer-membrane proteins of Yersinia pestis.

The yersinial plasmid-encoded outer-membrane proteins (Yops) are encoded by the low-Ca++ response virulence plasmid present in the yersiniae pathogenic for humans. It has been shown that in Yersinia pestis KIM there are 11 of these proteins, which are expressed maximally during growth at 37 degrees C in the absence of Ca++. To recover these proteins in outer membranes, it was necessary to transfer the Y. pestis low-Ca++ response plasmid to Yersinia pseudotuberculosis. However, two of these proteins (Yops M and N) were present in the culture medium after growth of Y. pestis; no other Yops were released by the bacteria. This raises the possibility of multiple roles for Yops in the virulence of Y. pestis.

Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment.

A procedure is described for fractionating Yersinia grown in small cultures into inner and outer membranes and soluble cytoplasmic proteins. The procedure was applied to the three recognized species of the genus grown under conditions simulating mammalian intracellular fluid with respect to Ca2+ and Mg2+. These conditions are known to elicit the production of the plague virulence antigen V. Isolates capable of making this antigen were compared with virulence-antigen-negative derivatives by two-dimensional electrophoresis. The V antigen was localized to the soluble protein fraction as a peptide that comigrates with the major component of a specific immunoprecipitate. This peptide had an apparent molecular weight of 38,000 and was not found in either apparent molecular weight of 38,000 and was not found in either membrane fraction. The comparison of virulence antigen-producers and nonproducers of Y. pseudotuberculosis and Y. enterocolitica revealed large qualitative and quantitative differences in outer membrane protein patterns, whereas the same comparison for Y. pestis showed only minor differences. The complexity of changes in the various protein fractions corroborate data in the literature indicating that extensive physiological changes occur in virulent organisms cultivated under simulated intracellular conditions.

Localization in Yersinia pestis of peptides associated with virulence.

An avirulent guanine auxotroph of wild-type Yersinia pestis was used to select isogenic mutants lacking invasive determinants of virulence including V and W antigens (Vwa-), genetically linked fibrinolysin, coagulase, and pesticin activities (Pst-), and the capacity to absorb exogenous pesticin and pigments including hemin (Pgm-). After growth in environments known to favor expression of these factors by the parent, cells were converted to spheroplasts and disrupted to obtain preparations of cytoplasm; particulate matter was separated into inner and outer membranes by sucrose gradient centrifugation. Peptides present in these fractions were then solubilized and compared by two-dimensional polyacrylamide gel electrophoresis. Components unique to Vwa+ cells, including V antigen, were restricted to the cytoplasmic fraction. In contrast, peptides possibly corresponding to fibrinolysin and coagulase were located primarily within the outer membrane of the Pst+ parent; pesticin was not identified. Similarly, a major outer membrane peptide, possibly representing the pesticin and pigment receptor, was peculiar to the Pgm+ parent. Accordingly, two of the virulence factors examined (Pst+ and Pgm+) can interact directly with host cells or fluids by virtue of their location on the bacterial surface. The remaining cytoplasmic Vwa+ determinant remains a candidate for a regulatory system whose role in pathogenicity is expression of functions required for intracellular survival.

Differential effects of deletions in lcrV on secretion of V antigen, regulation of the low-Ca2+ response, and virulence of Yersinia pestis.

The Yersinia pestis V antigen is necessary for full induction of low-calcium response (LCR) stimulon virulence gene transcription, and it also is a secreted protein believed to have a direct antihost function. We made four nonpolar deletions in lcrV of Y. pestis to determine if secretion, regulation, and virulence functions could be localized within the V antigen (LcrV). Deletion of amino acids 25 to 40 caused secretion of LcrV to be decreased in efficiency; however, removal of residues 108 to 125 essentially abolished LcrV secretion. Neither mutation had a significant effect on LCR regulation. This showed that LcrV does not have to be secreted to have its regulatory effect and that the internal structure of V antigen is necessary for its secretion. Both mutants were avirulent in mice, showing that the regulatory effect of LcrV could be separated genetically from its virulence role and raising the possibility that residues 25 to 40 are essential for the virulence function. This study provides the best genetic evidence available that LcrV per se is necessary for the virulence of Y. pestis. The repressed LCR phenotype of a mutant lacking amino acids 188 to 207 of LcrV raised the possibility that the deleted region is necessary for regulation of LCR induction; however, this mutant LcrV was weakly expressed and may not have been present in sufficient amounts to have its regulatory effect. In double mutants containing this mutant lcrV and also lacking expression of known LCR negative regulators (LcrG, LcrE, and LcrH), full induction of the LCR occurred in the absence of functional LcrV, indicating that LcrV promotes induction not as an activator per se but rather by inhibiting negative regulators.

LcrV of Yersinia pestis enters infected eukaryotic cells by a virulence plasmid-independent mechanism.

Yersinia pestis is the causative agent of bubonic plague and possesses a set of plasmid-encoded, secretable virulence proteins termed LcrV and Yops which are essential for survival in mammalian hosts. Yops and LcrV are secreted by a type III mechanism (Ysc), and Yops are unidirectionally targeted into the cytosol of associated eukaryotic cells in a tissue culture infection model. LcrV is required for Yops targeting, and recent findings have revealed that it can localize to the bacterial surface; however, its fate in this infection model has not been investigated in detail. In this study, we compared the localization of LcrV to that of the targeted proteins YopE and YopM by immunoblot analysis of fractions of Yersinia-infected HeLa cultures or by laser-scanning confocal microscopy of infected monolayers. Both LcrV and YopE were secreted by contact-activated, extracellularly localized yersiniae and were targeted to the HeLa cell cytosol. Although a significant amount of LcrV partitioned to the culture medium (unlike YopE), this extracellular pool of LcrV was not the source of the LcrV that entered HeLa cells. Unlike targeting of YopE and YopM, targeting of LcrV occurred in the absence of a functional Ysc apparatus and other virulence plasmid (pCD1)-expressed proteins. However, the Ysc is necessary for LcrV to be released into the medium, and our recent work has shown that localization of LcrV on the bacterial surface requires the Ysc. These results indicate that two mechanisms exist for the secretion of LcrV by Y. pestis, both of which are activated by contact with eukaryotic cells. LcrV secreted by the Ysc reaches the bacterial surface and the surrounding medium, whereas the second is a novel, Ysc-independent pathway which results in localization of LcrV in the cytosol of infected cells but not the surrounding medium.

Stickiness to Glass: Circadian Changes in the Cell Surface of Chlamydomonas reinhardi.

Conditions were found in which Chlamydomonas reinhardi exhibits a circadian alteration of its cell surface, measured as ability to stick to glass. Under these same conditions the cells also show circadian rhythms of cell division and release of daughter cells. The three rhythmic phenomena were shown to have typical properties of rhythms controlled by the biological clock. The rhythm of stickiness was used to demonstrate that in a mixed culture containing two cell populations with natural periods differing by 2 to 3 hours, the cells did not mutally entrain each other and that this rhythm could be successfully applied in an enrichment procedure for mutants of the biological clock. Stickiness was shown to be independent of growth and motility of the cells and unaffected by red or far red illimination. Minimally sticking cells did not affect the sticking of maximally sticking cells in a mixed culture; nor was there a progressive increase in stickiness shown at the minimum from one cycle to the next in a pure culture. These results indicate that sticking probably is not mediated by long lived adhesive material or enzymes excreted into the medium. Several tests of the sensitivity of stickiness to replacement of the growth medium by distilled water or water containing various compounds suggest that ions might play an important role in the sticking reaction.

Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins.

Yersinia pestis, the causative agent of plague, has a virulence determinant called the low-Ca2+ response (Lcr+ phenotype) that confers on the bacterium Ca2+ dependence for growth at 37 degrees C and expression of V antigen. This virulence determinant is common to the three species of Yersinia and is mediated by Lcr plasmids (called pCD in Y. pestis). In this study, we generated insertions of Mu dI1(Ap lac) in pCD1 of Y. pestis KIM, screened for cells showing transcriptional regulation by Ca2+, and obtained inserts that define at least four pCD1 genes. Their patterns of transcription under different growth conditions closely paralleled the pattern of expression of the V antigen. We tested for expression of Lcr-specific yersinial outer membrane proteins (Yops) by the pCD1::Mu dI1(Ap lac) plasmids. Four of the inserts each eliminated expression of a different Yop; one of these Yops was unique to Y. pestis. Two of the insertions affecting Yops caused avirulence, and one caused strongly decreased virulence of Y. pestis in mice. These data indicate that Yops, like the V antigen, are virulence attributes regulated in the low-Ca2+ response.

LcrG, a secreted protein involved in negative regulation of the low-calcium response in Yersinia pestis.

The purpose of this study was to define the function of LcrG, the product of the first gene in the lcrGVHyopBD operon of the low-Ca(2+)-response (LCR) virulence plasmid of Yersinia pestis. We created a Y. pestis strain having an in-frame deletion in lcrG. This nonpolar mutant had an abnormal LCR growth phenotype: it was unable to grow at 37 degrees C in the presence of 2.5 mM Ca2+ ("Ca2+ blind") but was able to grow at 37 degrees C when 18 mM ATP was present. At 37 degrees C it failed to downregulate the expression and secretion of its truncated product (LcrG), V antigen, and YopM. All of these mutant properties were complemented by plasmids carrying normal lcrG. However, a nonpolar lcrE mutation and an lcrH mutation (both also causing a Ca(2+)-blind phenotype) were not complemented in this way. The Y. pestis parent strain expressed LcrG at 37 degrees C in the presence and absence of Ca2+ and transported it to the medium when Ca2+ was absent. We identified two LCR-regulated loci, lcrD and yscDEF, required for this transport. Complementation analysis of the Y. pestis lcrR strain previously shown to lack the expression of LcrG showed that the loss of LcrG but not of LcrR caused the Ca(2+)-blind phenotype of that mutant. Taken together, the results show that LcrG is a negative regulator of the LCR, perhaps functioning in Ca2+ sensing along with LcrE.

Multiple effects of lcrD mutations in Yersinia pestis.

Plasmid pCD1 of Yersinia pestis contains a low-calcium response stimulon responsible for the temperature- and calcium-regulated expression and secretion of proteins involved in virulence, which include the V antigen and Yops. We have previously shown that insertional inactivation of the bicistronic lcrDR operon abolished the calcium requirement for growth at 37 degrees C and reduced expression of the V antigen and Yops. In this study, we constructed and characterized three mutants having nonpolar lcrD deletions. All three mutants lost the two main low-calcium response properties: a calcium requirement for growth at 37 degrees C and strong expression of the V antigen and Yops. The effects on virulence gene expression occurred at both the levels of transcription and secretion. The growth, transcription, and secretion defects could be at least partially complemented for two of the lcrD mutants by providing lcrD in trans. A third mutant could not be complemented, and a plasmid carrying this mutation had a dominant negative effect over normal LcrD function. In the three mutants, the amount of mutant LcrD protein detectable in immunoblots was inversely related to the amount of complementation. Taken together, these data indicate that LcrD function involves the interaction of LcrD with another molecule.

Mutations in yscC, yscD, and yscG prevent high-level expression and secretion of V antigen and Yops in Yersinia pestis.

The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca(2+)-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degrees C in the absence of Ca2+. In this study, we constructed and characterized mutants with in-frame deletions in yscC, yscD, and yscG of the ysc operon that contains yscA through yscM. All three mutants lost the Ca2+ requirement for growth at 37 degrees c, expressed only basal levels of V antigen and YopM in the presence or absence of Ca2+, and failed to secrete these proteins to the culture supernatant. Overproduction of YopM in these mutants failed to restore YopM export, showing that the mutations had a direct effect on secretion. The protein products of yscC, yscD, and yscG were identified and localized by immunoblot analysis. YscC was localized to the outer membrane of Y. pestis, while YscD was found in the inner membrane. YscG was distributed equally between the soluble and total membrane fractions. Double mutants were characterized to assess where YscC and YscD act in low-Ca2+ response (LCR) regulation. lcrH::cat-yscC and lcrH::cat-yscD double mutants were constitutively induced for expression of V antigen and YopM; however, these proteins were not exported. This finding showed that the ysc mutations did not directly decrease induction of LCR stimulon genes. In contrast, lcrE-yscC, lcrG-yscC, lcrE-yscD, and lcrG-yscD double mutants as well as an lcrE-lcrD double mutant expressed only basal levels of V antigen and YopM and also failed to secrete these proteins to the culture supernatant. These results indicated that a functional LCR secretion system was necessary for high-level expression of LCR stimulon proteins in the lcrE and lcrG mutants but not in an lcrH::cat mutant. Possible models of regulation which incorporate these results are discussed.

Yersinia pestis YopM: thrombin binding and overexpression.

In previous studies, Yersinia pestis YopM has been shown through mutational analysis to be necessary for virulence in mice and found to have homology with the thrombin-binding domain of the platelet receptor GPIb alpha. In this study, YopM was purified and shown by dot blot and chemical cross-linking tests to bind to human alpha-thrombin. No cross-linked product could be detected when human prothrombin was incubated with YopM. As a functional test of thrombin binding, it was shown that native but not boiled YopM inhibits thrombin-induced aggregation of human platelets. Control tests showed that YopM did not inactivate the platelets themselves, nor was its effect a nonspecific consequence of its very acidic isoelectric point. Microsequencing of YopM revealed an intact N terminus, indicating that functional YopM is not processed at the N terminus or secreted by a mechanism involving a cleavable signal sequence. Further characterization was made of an interesting effect on yopM expression that had been noticed in a previous study. A 1.5-kb HaeIII subclone overexpressed YopM in both Y. pestis and Escherichia coli compared with a larger clone containing the 5.3-kb HindIII-F fragment. To search for a possible regulator of YopM expression, the HindIII-F fragment was sequenced, revealing several open reading frames and three large repeated sequences. Deletional analysis showed that these were not involved in regulation of yopM. The data implicated a DNA structure 5' to yopM in moderating yopM expression.

YopD of Yersinia pestis plays a role in negative regulation of the low-calcium response in addition to its role in translocation of Yops.

Yersinia pestis produces a set of virulence proteins (Yops and LcrV) that are expressed at high levels and secreted by a type III secretion system (Ysc) upon bacterium-host cell contact, and four of the Yops are vectorially translocated into eukaryotic cells. YopD, YopB, and YopK are required for the translocation process. In vitro, induction and secretion occur at 37 degrees C in the absence of calcium. LcrH (also called SycD), a protein required for the stability and secretion of YopD, had initially been identified as a negative regulator of Yop expression. In this study, we constructed a yopD mutation in both wild-type and secretion-defective (ysc) Y. pestis to determine if the lcrH phenotype could be attributed to the decreased stability of YopD. These mutants were constitutively induced for expression of Yops and LcrV, despite the presence of the secreted negative regulator LcrQ, demonstrating that YopD is involved in negative regulation, regardless of a functioning Ysc system. Normally, secretion of Yops and LcrV is blocked in the presence of calcium. The single yopD mutant was not completely effective in blocking secretion: LcrV was secreted equally well in the presence and absence of calcium, while there was partial secretion of Yops in the presence of calcium. YopD is probably not rate limiting for negative regulation, as increasing levels of YopD did not result in decreased Yop expression. Overexpression of LcrQ in the yopD mutant had no significant effect on Yop expression, whereas increased levels of LcrQ in the parent resulted in decreased levels of Yops. These results indicate that LcrQ requires YopD to function as a negative regulator.