Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and Can Reverse Cassette-Induced Polar Effects.

As obligate intracellular bacteria, Chlamydia spp. have evolved numerous, likely intricate, mechanisms to create and maintain a privileged intracellular niche. Recent progress in elucidating and characterizing these processes has been bolstered by the development of techniques enabling basic genetic tractability. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Similar to other bacteria, many chlamydial genes exist within polycistronic operons, raising the possibility of polar effects mediated by insertion cassettes. Indeed, FRAEM-mediated deletion of Chlamydia trachomatistmeA negatively impacts the expression of tmeB We have adapted FRAEM technology by employing a gfp-bla cassette flanked by loxP sites. Conditional expression of Cre recombinase in ChlamydiatmeA containing a floxed cassette resulted in deletion of the marker and restoration of tmeB expression.IMPORTANCEC. trachomatis infections represent a significant burden to human health. The ability to genetically manipulate Chlamydia spp. is overcoming historic confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation in Chlamydia spp. requires further development, including mechanisms to generate markerless gene disruption. We leveraged a stepwise Cre-lox approach to excise selection marker genes from a deleted gene locus. We found this process to be efficient, and the removal of extraneous elements resulted in the reversal of a negative polar effect on a downstream gene. This technique facilitates a more direct assessment of gene function and adds to the Chlamydia molecular toolbox by facilitating the deletion of genes within operons.

Fluorescence-Reported Allelic Exchange Mutagenesis Reveals a Role for Chlamydia trachomatis TmeA in Invasion That Is Independent of Host AHNAK.

Development of approaches to genetically manipulate Chlamydia is fostering important advances in understanding pathogenesis. Fluorescence-reported allelic exchange mutagenesis (FRAEM) now enables the complete deletion of specific genes in C. trachomatis L2. We have leveraged this technology to delete the coding sequences for a known type III effector. The evidence provided here indicates that CT694/CTL0063 is a virulence protein involved in chlamydial invasion. Based on our findings, we designate the gene product corresponding to ct694-ctl0063translocated membrane-associated effector A (TmeA). Deletion of tmeA did not impact development of intracellular chlamydiae. However, the absence of TmeA manifested as a decrease in infectivity in both tissue culture and murine infection models. The in vitro defect was reflected by impaired invasion of host cells. TmeA binds human AHNAK, and we demonstrate here that AHNAK is transiently recruited by invading chlamydiae. TmeA, however, is not required for endogenous AHNAK recruitment. TmeA also impairs AHNAK-dependent actin bundling activity. This TmeA-mediated effect likely does not explain impaired invasion displayed by the tmeA strain of Chlamydia, since AHNAK-deficient cells revealed no invasion phenotype. Overall, our data indicate the efficacy of FRAEM and reveal a role of TmeA during chlamydial invasion that manifests independently of effects on AHNAK.

New frontiers in type III secretion biology: the Chlamydia perspective.

Members of the order Chlamydiales comprise a group of exquisitely evolved parasites of eukaryotic hosts that extends from single-celled amoeba to mammals. The most notable are human pathogens and include the agent of oculogenital disease Chlamydia trachomatis, the respiratory pathogen C. pneumoniae, and the zoonotic agent C. psittaci. All of these species are obligate intracellular bacteria that develop within parasitophorous vesicles termed inclusions. This demanding lifestyle necessitates orchestrated entry into nonphagocytic cells, creation of a privileged intracellular niche, and subversion of potent host defenses. All chlamydial genomes contain the coding capacity for a nonflagellar type III secretion system, and this mechanism has arisen as an essential contributor to chlamydial virulence. The emergence of tractable approaches to the genetic manipulation of chlamydiae raises the possibility of explosive progress in understanding this important contributor to chlamydial pathogenesis. This minireview considers challenges and recent advances that have revealed how chlamydiae have maintained conserved aspects of T3S while exploiting diversification to yield a system that exerts a fundamental role in the unique biology of Chlamydia species.

Perforin-2 restricts growth of Chlamydia trachomatis in macrophages.

Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that preferentially infects epithelial cells. Professional phagocytes provide C. trachomatis only a limited ability to survive and are proficient killers of chlamydiae. We present evidence herein that identifies a novel host defense protein, perforin-2, that plays a significant role in the eradication of C. trachomatis during the infection of macrophages. Knockdown of perforin-2 in macrophages did not alter the invasion of host cells but did result in chlamydial growth that closely mirrored that detected in HeLa cells. C trachomatis L2, serovar B, and serovar D and C. muridarum were all equally susceptible to perforin-2-mediated killing. Interestingly, induction of perforin-2 expression in epithelial cells is blocked during productive chlamydial growth, thereby protecting chlamydiae from bactericidal attack. Ectopic expression of perforin-2 in HeLa cells, however, does result in killing. Overall, our data implicate a new innate resistance protein in the control of chlamydial infection and may help explain why the macrophage environment is hostile to chlamydial growth.

Disulfide bonding within components of the Chlamydia type III secretion apparatus correlates with development.

Chlamydia spp. exhibit a unique biphasic developmental cycle whereby infectious elementary bodies (EBs) invade host epithelial cells and differentiate into noninfectious, metabolically active reticulate bodies (RBs). EBs posses a unique outer envelope where rigidity is achieved by disulfide bonding among cysteine-rich envelope-associated proteins. Conversely, these disulfide bonds become reduced in RBs to accommodate vegetative growth, thereby linking the redox status of cysteine-rich envelope proteins with progression of the developmental cycle. We investigated the potential role of disulfide bonding within the chlamydial type III secretion system (T3SS), since activity of this system is also closely linked to development. We focused on structural components of the T3S apparatus that contain an unusually high number of cysteine residues compared to orthologs in other secretion systems. Nonreducing SDS-PAGE revealed that EB-localized apparatus proteins such as CdsF, CdsD, and CdsC form higher-order complexes mediated by disulfide bonding. The most dramatic alterations were detected for the needle protein CdsF. Significantly, disulfide bonding patterns shifted during differentiation of developmental forms and were completely reduced in RBs. Furthermore, at later time points during infection following RB to EB conversion, we found that CdsF is reoxidized into higher-order complexes. Overall, we conclude that the redox status of specific T3SS apparatus proteins is intimately linked to the developmental cycle and constitutes a newly appreciated aspect of functionally significant alterations within proteins of the chlamydial envelope.

Biochemical and localization analyses of putative type III secretion translocator proteins CopB and CopB2 of Chlamydia trachomatis reveal significant distinctions.

Chlamydia spp. are among the many pathogenic Gram-negative bacteria that employ a type III secretion system (T3SS) to overcome host defenses and exploit available resources. Significant progress has been made in elucidating contributions of T3S to the pathogenesis of these medically important, obligate intracellular parasites, yet important questions remain. Chief among these is how secreted effector proteins traverse eukaryotic membranes to gain access to the host cytosol. Due to a complex developmental cycle, it is possible that chlamydiae utilize a different complement of proteins to accomplish translocation at different stages of development. We investigated this possibility by extending the characterization of C. trachomatis CopB and CopB2. CopB is detected early during infection but is depleted and not detected again until about 20 h postinfection. In contrast, CopB2 was detectible throughout development. CopB is associated with the inclusion membrane. Biochemical and ectopic expression analyses were consistent with peripheral association of CopB2 with inclusion membranes. This interaction correlated with development and required both chlamydial de novo protein synthesis and T3SS activity. Collectively, our data indicate that it is unlikely that CopB serves as the sole chlamydial translocation pore and that CopB2 is capable of association with the inclusion membrane.

Evidence that CT694 is a novel Chlamydia trachomatis T3S substrate capable of functioning during invasion or early cycle development.

Chlamydia trachomatis is an obligate intracellular parasite, occupies a membrane-bound vacuole throughout development and is capable of manipulating the eukaryotic host by translocating effector molecules via a type III secretion system (T3SS). The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early cycle events. We present evidence here that the C. trachomatis protein CT694 represents an early cycle-associated effector protein. CT694 is secreted by the Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two-hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. Additionally, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibres that correlated with the ability of CT694 to interact with AHNAK. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis, and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development particular to this species.

Bioinformatic and biochemical evidence for the identification of the type III secretion system needle protein of Chlamydia trachomatis.

Chlamydia spp. express a functional type III secretion system (T3SS) necessary for pathogenesis and intracellular growth. However, certain essential components of the secretion apparatus have diverged to such a degree as to preclude their identification by standard homology searches of primary protein sequences. One example is the needle subunit protein. Electron micrographs indicate that chlamydiae possess needle filaments, and yet database searches fail to identify a SctF homologue. We used a bioinformatics approach to identify a likely needle subunit protein for Chlamydia. Experimental evidence indicates that this protein, designated CdsF, has properties consistent with it being the major needle subunit protein. CdsF is concentrated in the outer membrane of elementary bodies and is surface exposed as a component of an extracellular needle-like projection. During infection CdsF is detectable by indirect immunofluorescence in the inclusion membrane with a punctuate distribution adjacent to membrane-associated reticulate bodies. Biochemical cross-linking studies revealed that, like other SctF proteins, CdsF is able to polymerize into multisubunit complexes. Furthermore, we identified two chaperones for CdsF, termed CdsE and CdsG, which have many characteristics of the Pseudomonas spp. needle chaperones PscE and PscG, respectively. In aggregate, our data are consistent with CdsF representing at least one component of the extended Chlamydia T3SS injectisome. The identification of this secretion system component is essential for studies involving ectopic reconstitution of the Chlamydia T3SS. Moreover, we anticipate that CdsF could serve as an efficacious target for anti-Chlamydia neutralizing antibodies.

Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle.

The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.

A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin.

The obligate intracellular bacterium Chlamydia trachomatis rapidly induces its own entry into host cells. Initial attachment is mediated by electrostatic interactions to heparan sulfate moieties on the host cell, followed by irreversible binding to an unknown secondary receptor. This secondary binding leads to the recruitment of actin to the site of attachment, formation of an actin-rich, pedestal-like structure, and finally internalization of the bacteria. How chlamydiae induce this process is unknown. We have identified a high-molecular-mass tyrosine-phosphorylated protein that is rapidly phosphorylated on attachment to the host cell. Immunoelectron microscopy studies revealed that this tyrosine-phosphorylated protein is localized to the cytoplasmic face of the plasma membrane at the site of attachment of surface-associated chlamydiae. The phosphoprotein was isolated by immunoprecipitation with the antiphosphotyrosine antibody 4G10 and identified as the chlamydial protein CT456, a hypothetical protein with unknown function. The chlamydial protein (Tarp) appears to be translocated into the host cell by type III secretion because it is exported in a Yersinia heterologous expression assay. Phosphotyrosine signaling across the plasma membrane preceded the recruitment of actin to the site of chlamydial attachment and may represent the initial signal transduced from pathogen to the host cell. These results suggest that C. trachomatis internalization is mediated by a chlamydial type III-secreted effector protein.

Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early-cycle development.

The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole termed an inclusion. During its intracellular developmental cycle, C. trachomatis maintains this intracellular niche, presumably by expressing a type III secretion system, which deploys a set of host cell-interactive proteins including inclusion membrane-localized proteins termed Incs. Some Incs are expressed and secreted by 2 h (early cycle) after infection, whereas the expression of type III-specific genes is not detectable until 6-12 h (mid-cycle). To resolve this paradox, we investigated the presence of a type III apparatus on elementary bodies (EBs) that might function early in infection. We demonstrate the existence of the type III secretory apparatus by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and immunoblot analyses of purified EB extracts. Immunoblots using polyclonal antibodies specific for the core apparatus component CdsJ identified this protein in both EB and reticulate body (RB) extracts. Furthermore, CdsJ-specific signals were detected by immunoblot of whole infected-culture extracts and by indirect immunofluorescence of infected monolayers at times before the detection of cdsJ-specific message. Finally, expression of IncC, expressed by 2 h after infection during C. trachomatis infections, in Yersinia pseudotuberculosis resulted in its secretion via the Yersinia type III apparatus. Based on these data, we propose a model in which type III secretion pores are present on EBs and mediate secretion of early Incs and possible additional effectors. Mid-cycle expression of type III genes would then replenish secretion apparatus on vegetative RBs and serve as a source of secretion pores for subsequently formed EBs.

Inhibition of fusion of Chlamydia trachomatis inclusions at 32 degrees C correlates with restricted export of IncA.

Chlamydia trachomatis is an obligate intracellular bacterium that develops within a parasitophorous vacuole termed an inclusion. The inclusion is nonfusogenic with lysosomes but intercepts lipids from a host cell exocytic pathway. Initiation of chlamydial development is concurrent with modification of the inclusion membrane by a set of C. trachomatis-encoded proteins collectively designated Incs. One of these Incs, IncA, is functionally associated with the homotypic fusion of inclusions. Inclusions also do not fuse when cultures are multiply infected with C. trachomatis and cultivated at 32 degrees C. We obtained evidence linking these experimental observations by characterizing IncA localization in 32 degrees C cultures. Analysis of inclusions by light and transmission electron microscopy confirmed that HeLa cells infected with multiple C. trachomatis elementary bodies and cultivated at 32 degrees C for 24 h contained multiple, independent inclusions. Reverse transcriptase PCR and immunoblot analyses of C. trachomatis-infected HeLa cells demonstrated the presence of IncA at 24 h in 32 degrees C cultures. When parallel cultures were probed with IncA-specific antibodies in indirect immunofluorescence assays, IncA was detectable in intracellular chlamydiae but not within the inclusion membrane. In addition, analysis of purified reticulate bodies from 37 and 32 degrees C cultures showed that bacterium-associated pools of IncA are enriched in cultures grown at 32 degrees C. Microscopic observation of infected cells revealed that some vacuoles had fused by 48 h postinfection, and this finding was correlated with the detection of IncA in inclusion membranes by immunofluorescence microscopy. The data are consistent with a requirement for IncA in fusions of C. trachomatis inclusions and suggest that the effect of incubation at 32 degrees C is manifested by restricted export of IncA to the inclusion membrane.

Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism.

The medically significant, obligate intracellular pathogen Chlamydia trachomatis replicates within vacuoles termed inclusions. A developmental cycle is initiated after entry into a host cell and is manifested by the transformation of infectious elementary bodies (EBs) to larger, non-infectious reticulate bodies (RBs). Analysis of the C. trachomatis genome has revealed that chlamydiae possess genes that may encode a type III secretion apparatus. In other Gram-negative pathogens, the type III secretion mechanism is used to target virulence factors directly to the host cell cytoplasm and is essential for full virulence. To evaluate the possibility of a functional type III secretion mechanism in C. trachomatis, we initially focused on a locus containing genes encoding products with similarity to chaperones (Scc1), secretion pore components (Cds1 and Cds2) and secreted proteins (CopN) from other type III systems. Gene expression was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA extracted from infected HeLa cell monolayers at 2, 6, 12 and 20 h after infection and normalized for the number of C. trachomatis genomes present. Message was detected for Scc1 at all times, whereas message for all other tested genes was detected in significant amounts at 12 h and 20 h. Immunoblot analysis with Scc1- and CopN-specific antibodies revealed that CopN and Scc1 were present in EBs, RBs and whole-culture extracts harvested 20 h after infection. CopN is homologous to the secreted protein YopN of Yersinia sp., and analysis of monolayers 20 h after infection via indirect immunofluorescence showed specific labelling of inclusion membranes when probed with CopN-specific antibodies but not with Scc1-specific antibodies. His-tagged CopN and a chlamydial cytoplasmic control protein (NrdB) were expressed in Yersinia enterocolitica containing or lacking the virulence plasmid pYV. CopN, but not NrdB, was secreted by Y. enterocolitica in a Ca2+- and pYV-dependent fashion. These data indicate that components of the putative type III apparatus of C. trachomatis are expressed and that at least one of these products is secreted by chlamydiae to the inclusion membrane. The observation that CopN is also secreted by the Yersinia type III apparatus provides support for the notion that chlamydiae secrete proteins via a type III mechanism.

Three temporal classes of gene expression during the Chlamydia trachomatis developmental cycle.

The obligate intracellular bacterium Chlamydia trachomatis has a unique developmental cycle that involves functionally and morphologically distinct cell types adapted for extracellular survival and intracellular multiplication. Infection is initiated by an environmentally resistant cell type called an elementary body (EB). Over the first several hours of infection, EBs differentiate into a larger replicative form, termed the reticulate body (RB). Late in the infectious process, RBs asynchronously begin to differentiate back to EBs, which accumulate within the lumen of the inclusion until released from the host cell for subsequent rounds of infection. In an effort to characterize temporal gene expression in relation to the chlamydial developmental cycle, we have used quantitative-competitive polymerase chain reaction (QC-PCR) and reverse transcription (RT)-PCR techniques. These analyses demonstrate that C. trachomatis double their DNA content every 2-3 h, with synthesis beginning between 2 and 4 h after infection. We determined the onset of transcription of specific temporal classes of developmentally expressed genes. RT-PCR analysis was performed on several genes encoding key enzymes or components of essential biochemical pathways and functions. This comparison encompassed approximately 8% of open reading frames on the C. trachomatis genome. In analysis of total RNA samples harvested at 2, 6, 12 and 20 h after infection, using conditions under which a single chlamydial transcript per infected cell is detected, three major temporal classes of gene expression were resolved. Initiation of transcription appears to occur in three temporal classes which we have operationally defined as: early, which are detected by 2 h after infection during the germination of EBs to RBs; mid-cycle, which appear between 6 and 12 h after infection and represent transcripts expressed during the growth and multiplication of RBs; or late, which appear between 12 and 20 h after infection and represent those genes transcribed during the terminal differentiation of RBs to EBs. Collectively, the data suggest that chlamydial early gene functions are weighted toward initiation of macromolecular synthesis and the establishment of their intracellular niche by modification of the inclusion membrane. Surprisingly, representative enzymes of intermediary metabolism and structural proteins do not appear to be transcribed until 10-12 h after infection; coinciding with the onset of observed binary fission of RBs. Late gene functions appear to be predominately those associated with the terminal differentiation of RBs back to EBs.

Skin breakthroughs in the year 2000.

Aging skin is a factor of many things-genetics, sun exposure, environmental insults, stress, and more. The signs of aging skin and cancer will be briefly reviewed followed by a discussion of current advances in dermatology, beginning with the new FDA regulation of sunscreens, the current state of tretinoin (Renova) and its future uses, the current state of antioxidants in the skin, and the latest therapies of microdermabrasion, neodymium:YAG lasers, and botulinum toxin.

Virulence role of V antigen of Yersinia pestis at the bacterial surface.

Yersinia pestis, the etiologic agent of plague, secretes a set of environmentally regulated, plasmid pCD1-encoded virulence proteins termed Yops and V antigen (LcrV) by a type III secretion mechanism (Ysc). LcrV is a multifunctional protein that has been shown to act at the level of secretion control by binding the Ysc inner-gate protein LcrG and to modulate the host immune response by altering cytokine production. LcrV also is essential for the unidirectional targeting of Yops to the cytosol of infected eukaryotic cells. In this study, we constructed an in-frame deletion within lcrG (DeltalcrG3) to further analyze the requirement of LcrV in Yop targeting. We confirmed the essentiality of LcrV and found that LcrG may have a facilitative role, perhaps by promoting efficient secretion of LcrV. We also constructed mutants of lcrV expressing LcrV truncated at the N or C terminus. Both the N and C termini of LcrV were required for the secretion of LcrV into the medium and targeting of Yops. LcrV was detected in punctate zones on the surface of fixed Y. pestis by laser-scanning confocal microscopy, and this localization required a functional Ysc. However, the truncated LcrV proteins were not found on the bacterial surface. Finally, we tested the ability of LcrV-specific Fab antibody fragments or full-length antibody to interfere with Yop targeting and found no interference, even though this antibody protects mice against plague. These results indicate that LcrV may function in Yop targeting at the extracellular surface of yersiniae and that the protective efficacy of LcrV-specific antibodies can be manifested without blocking Yop targeting.

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.

The V antigen of Yersinia pestis regulates Yop vectorial targeting as well as Yop secretion through effects on YopB and LcrG.

Yersinia pestis expresses a set of secreted proteins called Yops and the bifunctional LcrV, which has both regulatory and antihost functions. Yops and LcrV expression and the activity of the type III mechanism for their secretion are coordinately regulated by environmental signals such as Ca2+ concentration and eukaryotic cell contact. In vitro, Yops and LcrV are secreted into the culture medium in the absence of Ca2+ as part of the low-Ca2+ response (LCR). The LCR is induced in a tissue culture model by contact with eukaryotic cells that results in Yop translocation into cells and subsequent cytotoxicity. The secretion mechanism is believed to indirectly regulate expression of lcrV and yop operons by controlling the intracellular concentration of a secreted negative regulator. LcrG, a secretion-regulatory protein, is thought to block secretion of Yops and LcrV, possibly at the inner face of the inner membrane. A recent model proposes that when the LCR is induced, the increased expression of LcrV yields an excess of LcrV relative to LcrG, and this is sufficient for LcrV to bind LcrG and unblock secretion. To test this LcrG titration model, LcrG and LcrV were expressed alone or together in a newly constructed lcrG deletion strain, a delta lcrG2 mutant, of Y. pestis that produces low levels of LcrV and constitutively expresses and secretes Yops. Overexpression of LcrG in this mutant background was able to block secretion and depress expression of Yops in the presence of Ca2+ and to dramatically decrease Yop expression and secretion in growth medium lacking Ca2+. Overexpression of both LcrG and LcrV in the delta lcrG2 strain restored wild-type levels of Yop expression and Ca2+ control of Yop secretion. Surprisingly, when HeLa cells were infected with the delta lcrG2 strain, no cytotoxicity was apparent and translocation of Yops was abolished. This correlated with an altered distribution of YopB as measured by accessibility to trypsin. These effects were not due to the absence of LcrG, because they were alleviated by restoration of LcrV expression and secretion alone. LcrV itself was found to enter HeLa cells in a nonpolarized manner. These studies supported the LcrG titration model of LcrV's regulatory effect at the level of Yop secretion and revealed a further role of LcrV in the deployment of YopB, which in turn is essential for the vectorial translocation of Yops into eukaryotic cells.

Failure to detect binding of LcrH to the V antigen of Yersinia pestis.

V antigen of Yersinia pestis has been reported to bind the chaperone LcrH. We were unable to demonstrate this interaction. Despite methodological differences between our study and an earlier study, we believe that the previous findings were artifactual. One likely confounding element was the tendency of LcrH to adhere on its own to metal chelation chromatographic resin.

A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis.

The causative agent of plague, Yersinia pestis, contains a 75-kb plasmid, pCD1, which carries a virulence-related stimulon called the low-Ca2+ response stimulon (LCRS). LCRS operons are regulated by the environmental signals of temperature and Ca2+. This study characterized a portion of the lcrB region of pCD1, known to contain at least one gene necessary for the regulation of LCRS operons by Ca2+. The sequence of a 2-kb region revealed three open reading frames, designated yscQ, yscR, and yscS, predicted to encode acidic proteins of 34.4, 24.4, and 8.5 kDa. All three proteins were homologous to proteins involved in flagellar function or virulence. An antipeptide antibody specific for YscR was used to localize YscR to the inner membrane of Y. pestis. Analysis of yscR-phoA fusions supported a model for yscR which predicts four transmembrane regions and a large, central hydrophilic domain. In-frame deletion mutations of yscQ and yscR were constructed and moved into Y. pestis. Both mutants failed to show the restriction of growth that normally accompanies maximal LCRS induction. Unlike the parent Y. pestis, the yscR mutant did not respond to the absence of Ca2+ by increasing the net transcription or translation of the LCRS-encoded V antigen, YopM, or LcrG. The yscR mutant also was defective for secretion of V antigen, YopM, and LcrG. These findings implicate a dual role for YscR in regulation of LCRS operons and secretion of LCRS proteins and add to the developing picture of how secretion of virulence proteins may be coupled to transcriptional regulation in yersiniae.