Deborah M. Anderson

Two independent type III secretion mechanisms for YopE in Yersinia enterocolitica.

Pathogenic Yersinia species escape the infected host’s defense mechanisms by targeting cytotoxic Yop proteins into the cytoplasm of macrophages via a type III secretion pathway. Two separate secretion signals contained in YopE were identified, each of which were sufficient but not necessary for the secretion of reporter molecules. One signal is located within the coding sequence of the first 15 amino acids and is sufficient for the secretion of fusion proteins but not required for YopE secretion. The second signal is located downstream at residues 15–100 of YopE and is only recognized by the type III machinery when it is bound to SycE. We propose the existence of two independent mechanisms that allow for the secretion of Yop proteins.

Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one‐step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE targeting proceeds as a direct translocation of polypeptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for translocation into the eukaryotic cytosol. Here, we used the selective solubilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, YopM and YopN were found in the eukaryotic cytosol but not in the extracellular medium. When bound to SycE chaperone in the Yersinia cytoplasm, YopE residues 1–100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis failed to detect an extracellular intermediate of YopE targeting, suggesting a one‐step translocation mechanism.

Yersinia enterocolitica type III secretion: an mRNA signal that couples translation and secretion of YopQ

Pathogenic Yersinia species export Yop proteins via a type III machinery to escape their phagocytic killing during animal infections. Here, we reveal the type III export mechanism of YopQ. In the presence of calcium, when type III secretion was blocked, yopQ mRNA was not translated. The signal of YopQ sufficient for the secretion of translationally fused reporter proteins was contained within the first 10 codons of its open reading frame. Some frameshift mutations that completely altered the peptide sequence specified by this signal did not impair secretion of the reporter protein. Exchanging the upstream untranslated mRNA leader of yopQ for that of E. coli lacZ also did not affect secretion. However, removal of the first 15 codons abolished YopQ export. Pulse‐labelled YopE, but not YopQ, could be secreted after the polypeptide had been synthesized within the cytoplasm of Yersinia (post‐translational secretion). Thus, YopQ appears to be exported by a mechanism that couples yopQ mRNA translation with the type III secretion of the encoded polypeptide.

YopD and LcrH Regulate Expression of Yersinia enterocolitica YopQ by a Posttranscriptional Mechanism and Bind to yopQ RNA

Pathogenic yersiniae secrete 14 Yop proteins via the type III pathway. Synthesis of YopQ occurs when the type III machinery is activated by a low-calcium signal, but not when the calcium concentration is above 100 microM. To characterize the mechanism that regulates the expression of yopQ, mutants that permit synthesis of YopQ in the presence of calcium were isolated. Yersiniae bearing deletion mutations in yopN, tyeA, sycN, or yscB synthesized and secreted YopQ in both the presence and the absence of calcium. In contrast, yersiniae with a deletion in yopD or lcrH synthesized YopQ in the presence of calcium but did not secrete the polypeptide. These variants displayed no defect in YopQ secretion under low-calcium conditions, revealing that yopD and lcrH are required for the regulation of yopQ expression. Experiments with transcriptional and translational fusions to the npt reporter gene suggest that yopD and lcrH regulate yopQ expression at a posttranscriptional step. YopD and LcrH form a complex in the bacterial cytosol and bind yopQ mRNA. Models that can account for posttranscriptional regulatory mechanisms of yop expression are discussed.

Immunogenicity and Protective Immunity against Bubonic Plague and Pneumonic Plague by Immunization of Mice with the Recombinant V10 Antigen, a Variant of LcrV

ABSTRACT In contrast to Yersinia pestis LcrV, the recombinant V10 (rV10) variant (lacking residues 271 to 300) does not suppress the release of proinflammatory cytokines by immune cells. Immunization with rV10 generates robust antibody responses that protect mice against bubonic plague and pneumonic plague, suggesting that rV10 may serve as an improved plague vaccine.

Type III machines of Gram-negative pathogens: injecting virulence factors into host cells and more

Many Gram-negative bacteria that cause disease in either mammals or plants share a strategy of delivering toxic proteins into the cytoplasm of host cells known as type III secretion. Recent advances have provided a glimpse at the molecular nature of these lethal injection machines. Several groups have reported fibrous structures on bacterial surfaces that appear to be extensions of type III machines and necessary for toxin injection into host cells. Other research revealed complex mechanisms of secretion substrate recognition that presumably function to direct toxins to different locations during infection.

Pneumonic plague pathogenesis and immunity in Brown Norway rats.

The Brown Norway rat was recently described as a bubonic plague model that closely mimics human disease. We therefore evaluated the Brown Norway rat as an alternative small animal model for pneumonic plague and characterized both the efficacy and potency of vaccine candidates. When infected by intranasal instillation, these rats rapidly developed fatal pneumonic plague within 2 to 4 days of infection. Plague disease was characterized by severe alveolar edema and vascular hemorrhage in the lung in addition to fulminant necrotizing pneumonia caused by massive bacterial replication and inflammation. Twenty-four hours before death, animals developed systemic disease with an apparent delayed inflammatory response. We evaluated the ability of the protective antigen, LcrV, and a mutant derivative, V10, to protect these rats from pneumonic plague. Both were highly effective vaccines because complete protection was observed at challenge doses of 7500 LD(50). Antibody analyses suggested stronger potency of V10 immune sera compared with LcrV in the passive transfer of immunity to bubonic plague, with multiple neutralizing epitopes in LcrV. Taken together, these data demonstrate the effectiveness of inhibiting type III secretion in the prevention of pneumonic plague in rats and reveal critical contributions from both the cellular and humoral immune systems. Thus, the Brown Norway rat is an appealing alternative small animal model for the study of pneumonic plague pathogenesis and immunity.

Expression hierarchy in the Yersinia type III secretion system established through YopD recognition of RNA

The Yersinia type III secretion system (T3SS) is environmentally responsive to enable its rapid induction upon contact with host cells and is necessary for Yersiniae to establish a replicative niche and cause disease. YopD, a translocator protein, represses the expression of T3SS genes until signalled by environmental cues, a mechanism known as the low calcium response. In this work, we investigated recognition of target genes by Yersinia pestis YopD. Expression of all genes of the T3SS was induced in a yopD mutant, though not to the same degree, with effector Yops most affected. Two, short AU‐rich sequence elements up‐ and downstream of start codons of target genes were necessary but not sufficient for YopD mediated repression. Purified YopD–LcrH bound specifically to target RNAs in vitro with different relative affinities, with effector Yops having greater affinity. Together, the data suggest YopD binds to T3SS transcripts where it may prevent ribosome binding causing accelerated mRNA degradation. This regulatory mechanism may ensure an expression hierarchy during the low calcium response as low affinity YopD targets such as chaperones would be translated prior to high affinity targets such as effector Yops allowing the bacteria another layer of control over Yop translocation during infection.

Host Defense and the Airway Epithelium: Frontline Responses That Protect against Bacterial Invasion and Pneumonia

Airway epithelial cells are the first line of defense against invading microbes, and they protect themselves through the production of carbohydrate and protein matrices concentrated with antimicrobial products. In addition, they act as sentinels, expressing pattern recognition receptors that become activated upon sensing bacterial products and stimulate downstream recruitment and activation of immune cells which clear invading microbes. Bacterial pathogens that successfully colonize the lungs must resist these mechanisms or inhibit their production, penetrate the epithelial barrier, and be prepared to resist a barrage of inflammation. Despite the enormous task at hand, relatively few virulence factors coordinate the battle with the epithelium while simultaneously providing resistance to inflammatory cells and causing injury to the lung. Here we review mechanisms whereby airway epithelial cells recognize pathogens and activate a program of antibacterial pathways to prevent colonization of the lung, along with a few examples of how bacteria disrupt these responses to cause pneumonia.

Amino acid residues 196-225 of LcrV represent a plague protective epitope.

LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196-225 were necessary and sufficient for MAb BA5 binding. Compared to full-length LcrV, a variant lacking its residues 196-225 retained the ability of eliciting plague protection. These results identify LcrV residues 196-225 as a linear epitope that is recognized by the murine immune system to confer plague protection.