Ail confers serum resistance in humans and is a critical virulence factor of Y. pestis, the causative agent of plague. Here, the contribution of Ail for Y. pestis survival in the flea vector was ...examined. Rat or human but not mouse sera were bactericidal against a Y. pestis Δ
mutant at 28°C
. Complement components deposited rapidly on the Y. pestis surface as measured by immunofluorescent microscopy. Ail reduced the amount of active C3b on the Y. pestis surface. Human sera retained bactericidal activity against a Y. pestis Δ
mutant in the presence of mouse sera. However, in the flea vector, the serum protective properties of Ail were not required. Flea colonization studies using murine sera and Y. pestis KIM6
wild type, a Δ
mutant, and the Δ
control showed no differences in bacterial prevalence or numbers during the early stage of flea colonization. Similarly, flea studies with human blood showed Ail was not required for serum resistance. Finally, a variant of Ail (Ail
) from a human serum-sensitive Y. pestis subsp. microtus bv. Caucasica 1146 conferred resistance to human complement when expressed in the Y. pestis KIM6
Δ
mutant. This indicated that Ail activity was somehow blocked, most likely by lipooligosaccharide, in this serum sensitive strain.
This work contributes to our understanding of how highly virulent Y. pestis evolved from its innocuous enteric predecessor. Among identified virulence factors is the attachment invasion locus protein, Ail, that is required to protect Y. pestis from serum complement in all mammals tested except mice. Murine sera is not bactericidal. In this study, we asked, is bactericidal sera from humans active in Y. pestis colonized fleas? We found it was not. The importance of this observation is that it identifies a protective niche for the growth of serum sensitive and nonsensitive Y. pestis strains.
This review updates recent findings about Escherichia coli O157:H7 virulence factors and its bovine reservoir. This Shiga toxin (Stx)-producing E. coli belongs to the Enterohemorrhagic E. coli (EHEC) ...pathotype causing hemorrhagic colitis. Its low infectious dose makes it an efficient, severe, foodborne pathogen. Although EHEC remains in the intestine, Stx can translocate systemically and is cytotoxic to microvascular endothelial cells, especially in the kidney and brain. Disease can progress to life-threatening hemolytic uremic syndrome (HUS) with hemolytic anemia, acute kidney failure, and thrombocytopenia. Young children, the immunocompromised, and the elderly are at the highest risk for HUS. Healthy ruminants are the major reservoir of EHEC and cattle are the primary source of human exposure.
Advances in understanding E. coli O157:H7 pathogenesis include molecular mechanisms of virulence, bacterial adherence, type three secretion effectors, intestinal microbiome, inflammation, and reservoir maintenance.
Many aspects of E. coli O157:H7 disease remain unclear and include the role of the human and bovine intestinal microbiomes in infection. Therapeutic strategies involve controlling inflammatory responses and/or intestinal barrier function. Finally, elimination/reduction of E. coli O157:H7 in cattle using CRISPR-engineered conjugative bacterial plasmids and/or on-farm management likely hold solutions to reduce infections and increase food safety/security.
Intracellular Salmonella use a type III secretion system (TTSS) to translocate effector proteins across the phagosome membrane and thus promote vacuole membrane tubulation, resulting in intracellular ...survival. This work demonstrates that the effector SseJ binds the eukaryotic lipid transporter oxysterol binding protein 1 (OSBP1). SseJ directs OSBP1 to the endosomal compartment in a manner dependent on the TTSS located on Salmonella pathogenicity island 2 (SPI2). OSBP1 localization is mediated by both SseJ and another OSBP1-binding SPI2 translocated effector, the deubiquitinase SseL. Deletion of both SseJ and SseL reduced vacuolar integrity with increased bacteria released into the eukaryotic cytoplasm of epithelial cells, indicating that their combined activities are necessary for vacuole membrane stability. Cells knocked down for OSBP1 or deleted for the OSBP1-binding proteins VAPA/B also demonstrate loss of vacuole integrity, consistent with the hypothesis that OSBP1 recruitment is required for SPI2-mediated alterations that promote vacuolar integrity of salmonellae.
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•Salmonella-secreted effector SseJ binds eukaryotic oxysterol binding protein 1•SseJ directs OSBP1 to the endosomal compartment•OSBP1 localization is mediated by both SseJ and another effector, SseL•Deletion of both SseJ and SseL increases bacterial release into the cytoplasm
Kolodziejek et al. demonstrate that the Salmonella-secreted effector SseJ binds eukaryotic oxysterol binding protein 1 (OSBP1). SseJ directs OSBP1 to the endosome in concert with another effector, SseL. Deletion of both SseJ and SseL or OSBP1 knockdown increased bacterial cytoplasmic release, indicating that they are necessary for vacuolar stability.
Maintenance of phospholipid (PL) and lipopoly- or lipooligosaccharide (LPS or LOS) asymmetry in the outer membrane (OM) of Gram-negative bacteria is essential but poorly understood. The Yersinia ...pestis OM Ail protein was required to maintain lipid homeostasis and cell integrity at elevated temperature (37°C). Loss of this protein had pleiotropic effects. A Y. pestis
mutant and KIM6
wild type were systematically compared for (i) growth requirements at 37°C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernatants, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild-type phenotype. The
mutant grew normally at 28°C but lysed at 37°C when it entered stationary phase, as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immunofluorescence microscopy showed that the
mutant did not assemble Caf1 capsule. Expression of heat shock promoter
or
fused to a
operon reporter were not induced when the
mutant was shifted from 28°C to 37°C (
< 0.001 and
< 0.01, respectively). Mutant lysis was suppressed by addition of 11 mM glucose, 22 or 44 mM glycerol, 2.5 mM Ca
, or 2.5 mM Mg
to the growth medium or by a mutation in the phospholipase A gene (
::miniTn
,
, or PldA
). A model accounting for the temperature-sensitive lysis of the
mutant and the Ail-dependent stabilization of the OM tetraacylated LOS at 37°C is presented.
The Gram-negative pathogen Yersinia pestis transitions between a flea vector (ambient temperature) and a mammalian host (37°C). In response to 37°C, Y. pestis modifies its outer membrane (OM) by reducing the fatty acid content in lipid A, changing the outer leaflet from being predominantly hexaacylated to being predominantly tetraacylated. It also increases the Ail concentration, so it becomes the most prominent OM protein. Both measures are needed for Y. pestis to evade the host innate immune response. Deletion of
destabilizes the OM at 37°C, causing the cells to lyse. These results show that a protein is essential for maintaining lipid asymmetry and lipid homeostasis in the bacterial OM.
Pathogenic Yersinia have been a productive model system for studying bacterial pathogenesis. Hallmark contributions of Yersinia research to medical microbiology are legion and include: (i) the first ...identification of the role of plasmids in virulence, (ii) the important mechanism of iron acquisition from the host, (iii) the first identification of bacterial surface proteins required for host cell invasion, (iv) the archetypical type III secretion system, and (v) elucidation of the role of genomic reduction in the evolutionary trajectory from a fairly innocuous pathogen to a highly virulent species.
The outer membrane (OM) protein Ail (attachment invasion locus) was identified over 30 years ago as an invasin-like protein. Recent work on Ail continues to provide insights into Gram-negative pathogenesis. This review is a synopsis of the role of Ail in invasion, serum resistance, OM stability, thermosensing, and vaccine development.
Ail is shown to be an essential virulence factor with multiple roles in pathogenesis. The recent adaptation of Yersinia pestis to high virulence, which included genomic reduction to eliminate redundant protein functions, is a model to understand the emergence of new bacterial pathogens.
Summary
Salmonellae have the ability to invade, persist and replicate within an intracellular phagosome termed the Salmonella‐containing vacuole (SCV). Salmonellae alter lipid and protein content of ...the SCV membrane and manipulate cytoskeletal elements in contact with the SCV using the Salmonella pathogenicity island 1 (SPI‐2) type III secretion system effectors. These modifications result in microtubular‐based movement and morphological changes, which include endosomal tubulation of the SCV membrane. SseJ is a SPI‐2 effector that localizes to the cytoplasmic face of the SCV and esterifies cholesterol through its glycerophospholipid : cholesterol acyltransferase activity. SseJ enzymatic activity as well as localization to the SCV are determined by binding to the small mammalian GTPase, RhoA. This review will focus on current knowledge about the role of SseJ in SCV membrane modification and will discuss how the hypothesis that a major role of SPI‐2 effectors is to modify SCV protein and lipid content to promote bacterial intracellular survival.
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 x 10⁸ CFU of the ompX mutant survived, compared to the 50% lethal dose (LD₅₀) of 1.2 x 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.
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.
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844-3052, USA
Correspondence Gregory A. Bohach gbohach{at}uidaho.edu
The goal of this study was to ...characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis , Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6 + parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 °C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro . These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.
Abbreviations: C T , threshold cycle; HIS, heat-inactivated serum; normal human serum
These authors contributed equally to this work.
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