Scott A. Minnich

Thermoregulation in Yersinia enterocolitica is coincident with changes in DNA supercoiling

Yersinia enterocolitica is a facultative intracellular parasite, displaying the ability to grow saprophytically or invade and persist intracellularly in the mammalian reticuloendothelial system. The transition between such diverse environments requires the co‐ordinated regulation of specific sets of genes on both the chromosome and virulence plasmid. Temperature has a profound pleiotropic effect on gene expression and phenotypically promotes alterations in cell morphology, outer‐membrane protein synthesis, urease production, lipopolysaccharide synthesis, motility, and synthesis of genes involved in invasion of euKaryotic host cells. By examining thermoregulated flagella biosynthesis, we have determined that motility is repressed at 25° C (permissive temperature) with subinhibitory concentrations of novobiocin. These conditions also induce virulence gene expression suggesting novobiocin addition stimulates, at least partially, a high‐temperature environment. Furthermore, temperature‐shift experiments, using Y. enterocolitica containing pACYC184 as a reporter plasmid, indicate that thermo‐induced alterations of DNA supercoiling coincide with temperature‐induced phenotypic changes. A class of putative DNA gyrase mutant (novobiocin resistant) likewise demonstrates the 37° C phenotype when cultured at 25°C; it is non‐motile, urease negative, calcium growth dependent, and positive for Yop expression. These results support a model implicating DNA topology as a contributing factor of Y. enterocolitica thermoregulation.

Temperature-dependent regulation of Yersinia enterocolitica Class III flagellar genes

Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30°C and 37°C, Y. enterocolitica phase‐varies between motility and plasmid‐encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37°C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (σF) and flgM (anti‐σF), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar‐specific σ‐factor, σF, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti‐σF and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37°C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM− mutants, Y. enterocolitica flgM− mutants are fully virulent.

Co-ordinate, temperature-sensitive regulation of the three Yersinia enterocolitica flagellin genes.

Yersinia enterocolitica cells, when cultured at 30°C or below, are flagellated and motile. Cells cultured at 37°C or above lack flagella and are non‐motile. To identify flagellin genes that are a target of this temperature‐dependent regulation, a library of Y. enterocolitica genomic inserts in a phage λ vector was probed with the Salmonella typhimuriumfliC (flagellin) gene. A DNA fragment subcloned from a recombinant phage which hybridizes with the probe complements a non‐motile S. typhimuriumfliC−fljB− (flagellin‐minus) mutant. DNA sequence analysis shows that Y. enterocolitica contains three tandem flagellin genes, designated fleA, fleB and fleC. All three genes are co‐ordinately transcribed at low, but not high, temperature from fliA‐dependent (σF) promoters. Flagellin transcription arrests rapidly after upshift to 37°C (host temperature). In contrast, flagellin transcription resumes only after several generations when cells cultured at 37°C are downshifted to 28°C.

A 12-Base-Pair Deletion in the Flagellar Master Control Gene flhC Causes Nonmotility of the Pathogenic German Sorbitol-Fermenting Escherichia coli O157:H− Strains

An atypical, Stx2-producing, pathogenic Escherichia coli O157:H(-) strain has been isolated with increasing frequency from hemolytic uremic syndrome patients in Germany. The lack of the H7 antigen coupled with the strains ability to ferment sorbitol and express beta-glucuronidase have complicated its detection and identification. In this study, we have determined that the loss of motility in these German sorbitol-fermenting (SF) O157 strains is due to a 12-bp in-frame deletion in flhC that is required for transcriptional activation of genes involved in flagellum biosynthesis. Either complementation with a functional flhC or repair of this mutation restored H7 antigen expression and motility. PCR analysis of several nonmotile E. coli O157 strains from various geographical sources confirmed that the 12-bp flhC deletion is found only in the cluster of German SF O157 strains, providing a potentially useful marker by which these atypical strains can be identified. The loss of motility via mutations in the flhDC operon that we observed in the German SF O157 strains is consistent with a similar phenomenon currently observed in a significant subset of other important gram-negative pathogens.

Yersinia pestis two-component gene regulatory systems promote survival in human neutrophils

ABSTRACT Human polymorphonuclear leukocytes (PMNs, or neutrophils) are the most abundant innate immune cell and kill most invading bacteria through combined activities of reactive oxygen species (ROS) and antimicrobial granule constituents. Pathogens such as Yersinia pestis resist destruction by the innate immune system and are able to survive in macrophages and neutrophils. The specific molecular mechanisms used by Y. pestis to survive following phagocytosis by human PMNs are incompletely defined. To gain insight into factors that govern Y. pestis intracellular survival in neutrophils, we inactivated 25 two-component gene regulatory systems (TCSs) with known or inferred function and assessed susceptibility of these mutant strains to human PMN granule extracts. Y. pestis strains deficient for PhoPQ, KdpED, CheY, CvgSY, and CpxRA TCSs were selected for further analysis, and all five strains were altered for survival following interaction with PMNs. Of these five strains, only Y. pestis ΔphoPQ demonstrated global sensitivity to a panel of seven individual neutrophil antimicrobial peptides and serine proteases. Notably, Y. pestis ΔphoPQ was deficient for intracellular survival in PMNs. Iterative analysis with Y. pestis strains lacking the PhoP-regulated genes ugd and pmrK indicated that the mechanism most likely responsible for increased resistance to killing is 4-amino-4-deoxy-l-arabinose modification of lipid A. Together, the data provide new information about Y. pestis evasion of the innate immune system.

Outer Membrane Protein X (Ail) Contributes to Yersinia pestis Virulence in Pneumonic Plague and Its Activity Is Dependent on the Lipopolysaccharide Core Length

ABSTRACT Yersinia pestis, the causative agent of plague, is one of the most virulent microorganisms known. The outer membrane protein X (OmpX) in Y. pestis KIM is required for efficient bacterial adherence to and internalization by cultured HEp-2 cells and confers resistance to human serum. Here, we tested the contribution of OmpX to disease progression in the fully virulent Y. pestis CO92 strain by engineering a deletion mutant and comparing its ability in mediating pneumonic plague to that of the wild type in two animal models. The deletion of OmpX delayed the time to death up to 48 h in a mouse model and completely attenuated virulence in a rat model of disease. All rats challenged with 1 × 108 CFU of the ompX mutant survived, compared to the 50% lethal dose (LD50) of 1.2 × 103 CFU for the wild-type strain. Because murine serum is not bactericidal for the ompX mutant, the mechanism underlying the delay in time to death in mice was attributed to loss of adhesion/internalization properties but not serum resistance. The rat model, which is most similar to humans, highlighted the critical role of serum resistance in disease. To resolve conflicting evidence for the role of Y. pestis lipopolysaccharide (LPS) and OmpX in serum resistance, ompX was cloned into Escherichia coli D21 and three isogenic derivatives engineered to have progressively truncated LPS core saccharides. OmpX-mediated serum resistance, adhesiveness, and invasiveness, although dependent on LPS core length, displayed these functions in E. coli, independently of other Yersinia proteins and/or LPS. Also, autoaggregation was required for efficient OmpX-mediated adhesiveness and internalization but not serum resistance.

Characterization of an Escherichia coli O157:H7 O-Antigen Deletion Mutant and Effect of the Deletion on Bacterial Persistence in the Mouse Intestine and Colonization at the Bovine Terminal Rectal Mucosa

ABSTRACT Escherichia coli O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an E. coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the Δper mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the Δper mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the Δper mutant shed lower numbers of bacteria (P < 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the Δper mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (P < 0.05). Our previous work showed that flagella do not influence E. coli O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.

Neutrophils Are Resistant to Yersinia YopJ/P-Induced Apoptosis and Are Protected from ROS-Mediated Cell Death by the Type III Secretion System

Background The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined. Methodology/Principal Findings In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5. Conclusions Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.

A Rationale for Repression and/or Loss of Motility by Pathogenic Yersinia in the Mammalian Host

Pathogenic yersiniae either repress flagella expression under host conditions (Yersinia enterocolitica and Yersinia pseudotuberculosis) or have permanently lost this capability by mutation (Yersinia pestis). The block in flagella synthesis for the enteropathogenic Yersinia centers on fliA (sigmaF) repression. This repression ensures the downstream repression of flagellin structural genes which can be cross-recognized and secreted by virulence type III secretion systems. Y. pestis carries several flagellar mutations including a frame shift mutation in flhD, part of the flagellar master control operon. Repression of flagellins in the host environment may be critical because they are potent inducers of innate immunity. Artificial expression of flagellin in Y. enterocolitica completely attenuates virulence, supporting the hypothesis that motility is a liability in the mammalian host.

Yersinia pestis Ail: multiple roles of a single protein.

Yersinia pestis is one of the most virulent bacteria identified. It is the causative agent of plague—a systemic disease that has claimed millions of human lives throughout history. Y. pestis survival in insect and mammalian host species requires fine-tuning to sense and respond to varying environmental cues. Multiple Y. pestis attributes participate in this process and contribute to its pathogenicity and highly efficient transmission between hosts. These include factors inherited from its enteric predecessors; Y. enterocolitica and Y. pseudotuberculosis, as well as phenotypes acquired or lost during Y. pestis speciation. Representatives of a large Enterobacteriaceae Ail/OmpX/PagC/Lom family of outer membrane proteins (OMPs) are found in the genomes of all pathogenic Yersiniae. This review describes the current knowledge regarding the role of Ail in Y. pestis pathogenesis and virulence. The pronounced role of Ail in the following areas are discussed (1) inhibition of the bactericidal properties of complement, (2) attachment and Yersinia outer proteins (Yop) delivery to host tissue, (3) prevention of PMNL recruitment to the lymph nodes, and (4) inhibition of the inflammatory response. Finally, Ail homologs in Y. enterocolitica and Y. pseudotuberculosis are compared to illustrate differences that may have contributed to the drastic bacterial lifestyle change that shifted Y. pestis from an enteric to a vector-born systemic pathogen.