A Role for the SmpB-SsrA System in Yersinia pseudotuberculosis Pathogenesis

Yersinia utilizes a sophisticated type III secretion system to enhance its chances of survival and to overcome the host immune system. SmpB (small protein B) and SsrA (small stable RNA A) are components of a unique bacterial translational control system that help maintain the bacterial translational machinery in a fully operational state. We have found that loss of the SmpB-SsrA function causes acute defects in the ability of Yersinia pseudotuberculosis to survive in hostile environments. Most significantly, we show that mutations in smpB-ssrA genes render the bacterium avirulent and unable to cause mortality in mice. Consistent with these observations, we show that the mutant strain is unable to proliferate in macrophages and exhibits delayed Yop-mediated host cell cytotoxicity. Correspondingly, we demonstrate that the smpB-ssrA mutant suffers severe deficiencies in expression and secretion of Yersinia virulence effector proteins, and that this defect is at the level of transcription. Of further interest is the finding that the SmpB-SsrA system might play a similar role in the related type III secretion system that governs flagella assembly and bacterial motility. These findings highlight the significance of the SmpB-SsrA system in bacterial pathogenesis, survival under adverse environmental conditions, and motility

Kdo Hydrolase Is Required for Francisella tularensis Virulence and Evasion of TLR2-Mediated Innate Immunity

ABSTRACT The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD50) 2 log10 units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1β in a TLR2-dependent manner. In addition, TLR2−/− mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine

Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19

ABSTRACT Differences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background

Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host

Wild-Type and ΔBA Strains Display Different Patterns of Tissue Colonization

<p>Groups of seven mice per strain were infected via the orogastric route with 2 × 10⁹ cells and sacrificed on day 4 of infection. Harvested tissues were processed for CFU determination as described in Materials and Methods. Each symbol represents the CFU contained in the indicated tissue sample from one mouse. Data were analyzed by Student <i>t-</i>test in order to determine statistical significance of CFU counts in Peyer's Patches (<i>p</i> < 0.051), MLN (<i>p</i> < 0.271), and spleen (<i>p</i> < 0.044). The <i>smpB-ssrA</i> mutant was found to be able to reach extra-intestinal sites but unable to proliferate efficiently within the MLN and spleen.</p

Affect of <i>smpB-ssrA</i> Mutation on VirF and YmoA Protein Levels

<p>Cultures of wild-type (WT) or ΔBA strains of <i>Y. pseudotuberculosis</i> were grown in secretion non-permissive (lanes 1 and 2) or permissive medium (lanes 3 and 4) at 37 °C for 3.5 h as described in Materials and Methods. Bacteria were harvested and analyzed for VirF (A) and YmoA (B) proteins by Western blot analysis using specific polyclonal antibodies. Purified YmoA protein was used as a control (lane 5).</p

The ΔBA Mutant Has Impaired Motility

<div><p>(A) An equal number of cells from each bacterial strain were inoculated onto 0.25% agar-T medium plates and incubated for 48 h at room temperature.</p><p>(B) Wild-type and mutant cells were immobilized on grids, negatively stained with phosphotungstic acid, and analyzed using a JEOL JEM-1200EX II transmission electron microscope. Magnification = 12,000×; scale bar = 500 nm.</p><p>WT, wild type.</p></div

SmpB-SsrA Mediated Protein Tagging Activity Is Observed in <i>Y. pseudotuberculosis</i>

<div><p>(A) Schematic representation of the λ-cI-N-trpAt reporter construct encoded on the pKW540 plasmid and anticipated outcomes of protein tagging in wild-type (WT) or ΔBA strains.</p><p>(B) The λ-cI-N-trpAt reporter was induced in wild-type and mutant (ΔBA) strains, and protein samples were analyzed by Western blot using HRP-conjugated anti-H6 serum.</p><p>MW stds, protein molecular weight standards.</p></div

The ΔBA Mutant Strain Has Reduced Endogenous Levels of Effector Proteins

<p>Cultures of wild-type (WT) or ΔBA strains of <i>Y. pseudotuberculosis</i> were grown in low-calcium medium at 37 °C for 3.5 h. Bacteria were harvested and analyzed for endogenous levels of YopB, YopD, LcrV, LcrH, and YopE proteins by Western blot analysis using Yop-specific polyclonal antibodies.</p