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Pathogens that colonize deep tissues and spread systemically encounter the innate host resistance mechanism of complement-mediated lysis and complement opsonization leading to engulfment and degradation by phagocytic cells. Yersinia and Salmonella species have developed numerous strategies to block the antimicrobial effects of complement. These include recruitment of complement regulatory proteins factor H, C4BP, and vitronectin (Vn) as well as interference in late maturation events such as assembly of C9 into the membrane attack complex that leads to bacterial lysis. This review will discuss the contributions of various surface structures (proteins, lipopolysaccharide, and capsules) to evasion of complement-mediated immune clearance of the systemic pathogens Yersiniae and Salmonellae. Bacterial proteins required for recruitment of complement regulatory proteins will be described, including the details of their interaction with host regulatory proteins, where known. The potential role of the surface proteases Pla (Yersinia pestis) and PgtE (Salmonella species) on the activity of complement regulatory proteins will also be addressed. Finally, the implications of complement inactivation on host cell interactions and host cell targeting for type 3 secretion will be discussed., (© 2020 Federation of European Biochemical Societies.)

ToxR and TcpP, two winged helix-turn-helix (w-HTH) family transcription factors, co-activate expression of the toxT promoter in Vibrio cholerae. ToxT then directly regulates a number of genes required for virulence. In addition to co-activation of toxT, ToxR can directly activate the ompU promoter and repress the ompT promoter. Based on a previous study suggesting that certain wing residues of ToxR are preferentially involved in toxT co-activation compared to direct ompU activation, we employed alanine-scanning mutagenesis to determine which residues in the wing of ToxR are required for activation of each promoter. All of the ToxR wing residues tested that were critical for transcriptional activation of toxT and/or ompU were also critical for DNA binding. While some ToxR wing mutants had reduced interaction with TcpP, that reduced interaction did not correlate with a specific defect in toxT activation. Rather, such mutants also affected ompU activation and DNA binding. Based on these findings we conclude that the primary role of the wing of ToxR is to bind DNA, along with the DNA recognition helix of ToxR, and this function is required both for direct activation of ompU and co-activation of toxT., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: TcpP and ToxR coordinately regulate transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss of toxT activation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ∼50% toxT activation capacity. TcpP-C218S was also TcpH independent, since deletion of tcpH did not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression., Importance: The Vibrio cholerae transcription factor TcpP, in conjunction with ToxR, regulates transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

ToxR facilitates TcpP-mediated activation of the toxT promoter in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin secretion and toxin coregulated pilus expression. ToxR binds a region from -104 to -68 of the toxT promoter, from which ToxR recruits TcpP to the TcpP-binding site from -53 to -38. To precisely define the ToxR-binding site within the toxT promoter, promoter derivatives with single-base-pair transversions spanning the ToxR-footprinted region were tested for transcription activation and DNA binding. Nine transversions between -96 to -83 reduced toxT promoter activity 3-fold or greater, and all nine reduced the relative affinity of the toxT promoter for ToxR at least 2-fold, indicating that activation defects were due largely to reduced binding of ToxR to the toxT promoter. Nucleotides important for ToxR-dependent toxT activation revealed a consensus sequence of TNAAA-N(5)-TNAAA extending from -96 to -83, also present in other ToxR-regulated promoters. When these consensus nucleotides were mutated in the ompU, ompT, or ctxA promoters, ToxR-mediated regulation was disrupted. Thus, we have defined the core ToxR-binding site present in numerous ToxR-dependent promoters and we have precisely mapped the binding site for ToxR to a position three helical turns upstream of TcpP in the toxT promoter.

ToxR of Vibrio cholerae directly activates the ompU promoter, but requires a second activator, TcpP to activate the toxT promoter. ompU encodes a porin, while toxT encodes the transcription factor, ToxT, which activates V. cholerae virulence genes including cholera toxin and the toxin co-regulated pilus. Using an ompU-sacB transcriptional fusion, toxR mutant alleles were identified that encode ToxR molecules defective for ompU promoter activation. Many toxR mutants defective for ompU activation affected residues involved in DNA binding. Mutants defective for ompU activation were also tested for activation of the toxT promoter. ToxR-F69A and ToxR-V71A, both in the α-loop of ToxR, were preferentially defective for ompU activation, with ToxR-V71A nearly completely defective. Six mutants from the ompU-sacB selection showed more dramatic defects in toxT activation than ompU activation. All but one of the affected residues map to the wing domain of the winged helix-turn-helix of ToxR. Some ToxR mutants preferentially affecting toxT activation had partial DNA-binding defects, and one mutant, ToxR-P101L, had altered interactions with TcpP. These data suggest that while certain residues in the α-loop of ToxR are utilized to activate the ompU promoter, the wing domain of ToxR contributes to both promoter binding and ToxR/TcpP interaction facilitating toxT activation., (© 2011 Blackwell Publishing Ltd.)

Yersinia pestis genome sequencing projects have revealed six intact uncharacterized chaperone/usher systems with the potential to play roles in plague pathogenesis. We cloned each locus and expressed them in the Δfim Escherichia coli strain AAEC185 to test the assembled Y. pestis surface structures for various activities. Expression of each chaperone/usher locus gave rise to specific novel fibrillar structures on the surface of E. coli. One locus, y0561-0563, was able to mediate attachment to human epithelial cells (HEp-2) and human macrophages (THP-1) but not mouse macrophages (RAW264.7), while several loci were able to facilitate E. coli biofilm formation. When each chaperone/usher locus was deleted in Y. pestis, only deletion of the previously described pH 6 antigen (Psa) chaperone/usher system resulted in decreased adhesion and biofilm formation. Quantitative RT-PCR (qRT-PCR) revealed low expression levels for each novel chaperone/usher system in vitro as well as in mouse tissues following intravenous infection. However, a Y. pestis mutant in the chaperone/usher locus y1858-1862 was attenuated for virulence in mice via the intravenous route of infection, suggesting that expression of this locus is, at some stage, sufficient to affect the outcome of a plague infection. qRT-PCR experiments also indicated that expression of the chaperone/usher-dependent capsule locus, caf1, was influenced by oxygen availability and that the well-described chaperone/usher-dependent pilus, Psa, was strongly induced in minimal medium even at 28 °C rather than 37 °C, a temperature previously believed to be required for Psa expression. These data indicate several potential roles for the novel chaperone/usher systems of Y. pestis in pathogenesis and infection-related functions such as cell adhesion and biofilm formation.

ToxR-dependent recruitment of TcpP to the toxT promoter facilitates toxT transcription in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin expression and secretion. Although TcpP usually requires ToxR to activate the toxT promoter, TcpP overexpression can circumvent the requirement for ToxR in this process. To define nucleotides critical for TcpP-dependent promoter recognition and activation, a series of toxT promoter derivatives with single-base-pair transversions spanning the TcpP-binding site were generated and used as plasmid-borne toxT-lacZ fusions, as DNA mobility shift targets, and as allelic replacements of the chromosomal toxT promoter. When present in ΔtoxR V. cholerae overexpressing TcpP, several transversions affecting nucleotides within two direct repeats present in the TcpP-binding region (TGTAA-N(6)-TGTAA) caused defects in TcpP-dependent toxT-lacZ fusion activation and toxin production. Electrophoretic mobility shift assays demonstrated that these same transversions reduced the affinity of the toxT promoter for TcpP. The presence of ToxR suppressed transcription activation defects associated with most, but not all, transversions. Particularly, the central thymine nucleotide of both pentameric repeats was essential for efficient toxT activation, even in the presence of ToxR. These results suggest that the toxT promoter recognition function provided by ToxR can facilitate the interaction of TcpP with the toxT promoter but is insufficient for promoter activation when the TcpP-binding site has been severely compromised by mutation. Thus, the interaction of TcpP with nucleotides of the direct repeat sequences appears to be a prerequisite for toxT promoter activation.

Although adhesion to host cells is a critical step in the delivery of cytotoxic Yop proteins by Yersinia pestis, the mechanism has not been defined. To identify adhesins critical for Yop delivery, we initiated two transposon mutagenesis screens using the mariner transposon. To avoid redundant cell binding activities, we initiated the screen with a strain deleted for two known adhesins, pH 6 antigen and the autotransporter, YapC, as well as the Caf1 capsule, which is known to obscure some adhesins. The mutants that emerged contained insertions within the ail (attachment and invasion locus) gene of Y. pestis. A reconstructed mutant with a single deletion in the ail locus (y1324) was severely defective for delivery of Yops to HEp-2 human epithelial cells and significantly defective for delivery of Yops to THP-1 human monocytes. Specifically, the Yop delivery defect was apparent when cell rounding and translocation of an ELK-tagged YopE derivative into host cells were monitored. Although the ail mutant showed only a modest decrease in cell binding capacity in vitro, the KIM5 Deltaail mutant exhibited a >3,000-fold-increased 50% lethal dose in mice. Mice infected with the Deltaail mutant also had 1,000-fold fewer bacteria in their spleens, livers, and lungs 3 days after infection than did those infected with the parental strain, KIM5. Thus, the Ail protein is critical for both Y. pestis type III secretion in vitro and infection in mice.

YapC, a putative Yersinia pestis autotransporter protein, shows strong homology to the enterotoxigenic Escherichia coli adhesin TibA. As a potentially important surface protein of Y. pestis, we analysed YapC for several activities. When expressed in the non-pathogenic Fim(-) E. coli strain AAEC185, YapC mediated attachment to both murine-derived macrophage-like cells (RAW264.7) and human-derived epithelial-like cells (HEp-2). In addition, expression of YapC on the surface of E. coli led to autoaggregation in DMEM tissue culture medium, a phenomenon associated with virulence in Yersinia species. YapC also mediated formation of biofilm-like deposits by E. coli AAEC185. Deletion of yapC in Y. pestis strain KIM5 resulted in no change in adhesion to either RAW264.7 or HEp-2 cells, or in biofilm formation. Lack of a phenotype for the Y. pestis DeltayapC mutant may reflect the relatively low level of yapC expression in vitro, as assessed by RT-PCR, and/or redundant functions expressed in vitro. These data demonstrate several activities for YapC that may function during Y. pestis infection.

Expression of toxT, the transcription activator of cholera toxin and pilus production in Vibrio cholerae, is the consequence of a complex cascade of regulatory events that culminates in activation of the toxT promoter by TcpP and ToxR, two membrane-localized transcription factors. Both are encoded in operons with genes whose products, TcpH and ToxS, which are also membrane localized, are hypothesized to control their activity. In this study we analyzed the role of TcpH in controlling TcpP function. We show that a mutant of V. cholerae lacking TcpH expressed virtually undetectable levels of TcpP, although tcpP mRNA levels remain unaffected. A time course experiment showed that levels of TcpP, expressed from a plasmid, are dramatically reduced over time without co-overexpression of TcpH. By contrast, deletion of toxS did not affect ToxR protein levels. A fusion protein in which the TcpP periplasmic domain is replaced with that of ToxR remains stable, suggesting that the periplasmic domain of TcpP is the target for degradation of the protein. Placement of the periplasmic domain of TcpP on ToxR, an otherwise stable protein, results in instability, providing further evidence for the hypothesis that the periplasmic domain of TcpP is a target for degradation. Consistent with this interpretation is our finding that derivatives of TcpP lacking a periplasmic domain are more stable in V. cholerae than are derivatives in which the periplasmic domain has been truncated. This work identifies at least one role for the periplasmic domain of TcpP, i.e., to act as a target for a protein degradation pathway that regulates TcpP levels. It also provides a rationale for why the V. cholerae tcpH mutant strain is avirulent. We hypothesize that regulator degradation may be an important mechanism for regulating virulence gene expression in V. cholerae.

Virulence in Vibrio cholerae requires activation of toxT by two membrane-localized activators, TcpP and ToxR. We isolated 12 tcpP activation mutants that fell into two classes: class I mutants were inactive irrespective of the presence of ToxR, and class II mutants exhibited near wild-type activity when coexpressed with ToxR. Most class I mutants had lesions in the wing domain predicted by homology with the winged helix-turn-helix family of activators. Class I mutants bound promoter DNA poorly and were largely unable to interact with ToxR in a crosslinking assay, whereas class II mutants retained physical interaction with ToxR. One mutant constructed in vitro bound DNA poorly but nevertheless responded to ToxR by activating toxT and also maintained ToxR interaction. We propose that ToxR interaction, but not DNA binding, is essential for TcpP function and that the wing domain of TcpP enables contact with ToxR required for productive TcpP-RNA polymerase association.

ToxR is a bitopic membrane protein that controls virulence gene expression in Vibrio cholerae. Its cytoplasmic domain is homologous to the winged helix-turn-helix ('winged helix') DNA-binding/transcription activation domain found in a variety of prokaryotic and eukaryotic regulators, whereas its periplasmic domain is of ill-defined function. Several genes in V. cholerae are regulated by ToxR, but by apparently different mechanisms. Whereas ToxR directly controls the transcription of genes encoding two outer membrane proteins, OmpU and OmpT, it co-operates with a second membrane-localized transcription factor called TcpP to activate transcription of the gene encoding ToxT, which regulates transcription of cholera toxin (ctxAB) and the toxin-co-regulated pilus (tcp). To determine the requirements for gene activation by ToxR, different domains of the protein were analysed for their ability to control expression of toxT, ompU and ompT. Soluble forms of the cytoplasmic winged-helix domain regulated ompU and ompT gene expression properly but did not activate toxT transcription. Membrane localization of the winged helix was sufficient for both omp gene regulation and TcpP-dependent toxT transcription, irrespective of the type of periplasmic domain or even the presence of a periplasmic domain. These results suggest that (i) the major function for membrane localization of ToxR is for its winged-helix domain to co-operate with TcpP to activate transcription; (ii) the periplasmic domain of ToxR is not required for TcpP-dependent activation of toxT transcription; and (iii) membrane localization is not a strict requirement for DNA binding and transcription activation by ToxR.

ToxR is required in Vibrio cholerae for transcriptional activation of the toxT gene, the protein product of which activates numerous genes involved in virulence. Although ToxR cannot activate the toxT promoter in Escherichia coli, the products of the tcpPH operon are shown here to activate the toxT promoter, and co-expression with ToxRS enhances activation. An identical pattern was seen in a DeltatcpPDeltatoxR strain of V. cholerae when TcpPH or ToxRS was expressed from plasmids. Although overexpression of the TcpP/H proteins in V. cholerae partially complemented both a DeltatoxR strain and a DeltatcpPDeltatoxR double mutant for toxin production and toxT-lacZ activation, the presence of ToxR greatly increased their expression. Analysis of a toxT-lacZ promoter deletion series demonstrated that TcpP was able to interact functionally with the toxT promoter downstream of the ToxR binding site. This was confirmed using electrophoretic mobility shift assays of this toxT promoter deletion series and DNase I footprinting analysis, which showed that TcpP interacts with the promoter region from -51 to -32, whereas ToxR protected a region from -100 to -69. In addition, membranes containing endogenous levels of ToxR bound more readily to the toxT promoter than did membranes containing only TcpP. Characterization of a number of tcpP substitution mutants revealed one derivative (TcpP-H93L) that, when overexpressed, was markedly defective for toxT activation, cholera toxin and TcpA (toxin co-regulated pilus) production and DNA binding; however, toxT activation by TcpP-H93L was restored in the presence of ToxR, suggesting that ToxR can provide the promoter recognition function for toxT activation. Two additional mutant derivatives, TcpP-W68L and TcpP-R86A, failed to activate toxT or direct toxin and TcpA production in the presence or absence of ToxR. Both TcpP-W68L and TcpP-R86A, like TcpP-H93L, were defective for DNA binding. Finally, a ToxR mutant derivative, ToxR-G80S, served to separate the different roles of ToxR on different promoters. Although ToxR-G80S was inefficient at activating the ompU promoter in V. cholerae (ompU encodes an outer membrane porin regulated by ToxR), it was fully capable of activating the toxT promoter. These data suggest that ToxR is not a direct activator in the toxT expression system but, instead, enhances the activity of TcpP, perhaps by recruiting it to the toxT promoter under conditions in which expression levels of TcpP are too low for it to activate toxT efficiently on its own.

A highly conserved 14-amino-acid region of the chicken beta1 integrin chain (beta1chk residues 151-168) hypothesized to be involved in divalent cation coordination was analysed to determine whether invasin uses the same structural determinants as fibronectin (Fn) to recognize receptors. For the most part, both proteins required similar beta1 chain residues for integrin recognition, although the relative preference of the integrin for the two substrates could be inverted mutationally. Substitution mutations in the amino terminal residues of this region resulted in defective binding to both substrates by the receptor, while substitutions at the carboxyl-terminal end of this region were better tolerated. A derivative carrying the double substitution (KDD160RDV) had the unique phenotype of maintaining Fn binding while abolishing invasin binding, indicating that this region of the receptor may determine substrate specificity. These data indicate that the integrin beta1 chain possesses a ligand binding site shared by invasin and Fn that can be altered by mutation to allow greater preference for the normally lower affinity substrate Fn than for invasin. It was also established that the region analysed has the ability to bind divalent cations.

To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human alpha3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the alpha3 chain, alpha3-W220A, failed to promote adhesion to the natural alpha3 beta1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, alpha3-Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, alpha3-D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-alpha3 antibody, indicating that the second and third repeats of the alpha3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin alpha chain mutations, as alpha4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.

The Yersinia pseudotuberculosis invasin protein promotes bacterial uptake into normally non-phagocytic cells. Combinations of six alanine substitutions in a region of invasin previously shown to be important for bacterial internalization were analyzed using binomial and codon mutagenesis strategies. A single pool of mutants, potentially containing 64 derivatives with various combinations of alanine substitutions, was enriched by one passage through HEp2 cells. DNA was isolated from the resulting pool of internalization-competent bacteria and sequenced in a single set of reactions to determine which alanine substitutions maintained activity. Results of the single sequencing run performed on the pool indicated that strains harboring the D911A substitution were absent after enrichment, confirming the importance of an aspartate residue at this site. When single clones were subsequently isolated from the pool, those containing multiple alanine substitutions in invasin showed uptake defects that were additive, with the exception of S904A/M912A and S910A/M912A double mutants. Binomial mutagenesis combined with a pooled enrichment and sequencing strategy, called 'SWIM' mutagenesis (selection without isolation of mutants), could be applied to any system for which there exists an enrichment scheme, using a single oligonucleotide pool to analyze multiple residues.