Vulnerabilities in Yersinia pestis caf Operon Are Unveiled by a Salmonella Vector

During infection, Yersinia pestis uses its F1 capsule to enhance survival and cause virulence to mammalian host. Since F1 is produced in large quantities and secreted into the host tissues, it also serves as a major immune target. To hold this detrimental effect under proper control, Y. pestis expresses the caf operon (encoding the F1 capsule) in a temperaturedependent manner. However, additional properties of the caf operon limit its expression. By overexpressing the caf operon in wild-type Salmonella enterica serovar Typhimurium under a potent promoter, virulence of Salmonella was greatly attenuated both in vitro and in vivo. In contrast, expression of the caf operon under the regulation of its native promoter exhibited negligible impairment of Salmonellae virulence. In-depth investigation revealed all individual genes in the caf operon attenuated Salmonella when overexpressed. The deleterious effects of caf operon and the caf individual genes were further confirmed when they were overexpressed in Y. pestis KIM6+. This study suggests that by using a weak inducible promoter, the detrimental effects of the caf operon are minimally manifested in Y. pestis. Thus, through tight regulation of the caf operon, Y. pestis precisely balances its capsular anti-phagocytic properties with the detrimental effects of caf durin

Milk-based nutraceutical for treating autoimmune arthritis via the stimulation of IL-10- and TGF-β-producing CD39+ regulatory T cells.

Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39+ Tregs producing TGF-β and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I

Effect of overexpression of <i>caf</i> operon or individual <i>caf</i> genes on <i>Yersinia</i> antimicrobial and temperature susceptibilities.

<p>Six strains of KIM6+/pF1, pHF, pSA, pSF1, pSM, and pY were analyzed for their sensitivities to (<b>A</b>) erythromycin, (<b>B</b>) PMB, (<b>C</b>) hydrogen peroxide, (<b>D</b>) bile salt, (<b>E</b>) temperature of 37°C, and (<b>F</b>) bile salt combined with temperature of 37°C. (<b>A</b>, <b>B</b>) The erythromycin and PMB MICs of the 6 strains were respectively determined. (<b>C</b>) The 6 strains were incubated with 2.5 mM H₂O₂ for 1 hr and the survival percentages were determined in comparison with the non-treated controls, respectively. (<b>D</b>) The 6 strains were dropped onto BHI agar containing 1% bile and were incubated at 27°C for 48 hrs for CFU enumeration in comparison with those grown on BHI agar without bile, respectively. (<b>E</b>) The 6 strains were dropped onto BHI agar and were incubated at 37°C for CFU enumeration in comparison with those grown on BHI agar at 27°C, respectively. (<b>F</b>) The 6 strains were dropped onto BHI agar containing 1% bile and were incubated at 37°C for CFU enumeration in comparison with those grown on BHI agar without bile at 27°C, respectively. All experiments (<b>A</b> to <b>F</b>) were statistically analyzed for significant differences among these 6 strains using the Tukey Kramer multiple comparisons test with * <i>P</i><0.05, ** <i>P</i><0.01, and *** <i>P</i><0.001. Depicted (<b>A</b> to <b>F</b>) are the mean ± SEM of three independent experiments.</p

Effects of overexpression of F1 capsule on <i>Salmonella</i> phenotypes.

<p>(<b>A</b>) Overexpression of F1 capsule resultant in anti-agglutination phenotype of <i>Salmonella</i> bacilli. After centrifugation, P1-pF1 and -pY were pelleted to the tube bottom while -pHF was not. (<b>B</b>) Detection of F1 capsular expression by Western blotting. Strain P1-pF1 did not express detectable F1 capsular proteins until 12 hrs post-inoculation, but F1 capsular proteins were visible from -pHF as early as 4 hrs post-inoculation. Control P1-pY did not produce F1 capsule. (<b>C</b>) Quantification of F1 capsule. The F1 capsular protein yields of P1-pF1 and -pHF were determined and their differences were analyzed using the Tukey Kramer multiple comparisons test with ** <i>P</i><0.01. Depicted are the mean ± SEM of three independent experiments.</p

Expression of <i>E</i>. <i>coli cfaI</i> fimbrial operon in <i>Lactococcus lactis</i>.

<p><b>(A)</b> Schematic map for expression of <i>E</i>. <i>coli cfaI</i> in <i>L</i>. <i>lactis</i> (pBzMM153). The elements of the composite promoter are boxed. Each structural gene is fused in-frame to a different lactococcal secretion signal peptide, and each gene is followed by its own STOP codon. No other functional elements are interspersed except for one Shine-Dalgarno (SD) sequence upstream of a fusion between extracellular (Exp4) protein and <i>cfaA</i>. All (SD) sequences are marked. <b>(B)</b> CFA/I fimbriae expression in <i>L</i>. <i>lactis</i>. Lane1, molecular weight (MW) markers; lane 2, <i>Salmonella</i>-CFA/I (H696) strain; lane 3, <i>L</i>. <i>lactis</i> bearing the empty pMSP3535H3 vector; lanes 4,5, pBzMM156 (nisin-inducible promoter) clones 1 and 2; lanes 6,7, pBzMM155 (p23 promoter) clones 1 and 2; and lanes 8–10, pBzMM153 (synthetic composite promoter) clones 1–3. <b>(C, D)</b> Immunogold staining of <b>(C)</b><i>L</i>. <i>lactis</i> vector and <b>(D)</b><i>L</i>. <i>lactis</i>-CFA/I with anti-CFA/I antibody. Arrows point to gold particles on fimbrial structures projecting from the cell wall.</p

Schematic maps of plasmid pSMA, pSA, pSM, and pSF1 and growth rates of <i>Salmonella</i> harboring these plasmids.

<p>(<b>A</b>) pSMA was generated by deletion of <i>caf1</i> gene from pHF. (<b>B</b>) pSA was constructed by deletion of <i>caf1M</i> gene from pSMA. (<b>C</b>) pSM was created by deletion of <i>caf1A</i> gene from pSMA. (<b>D</b>) pSF1 was produced by cloning <i>caf1</i> gene from pF1 to pHF to replace the <i>caf1Mcaf1Acaf1</i> genes. The <i>caf1</i>(-), <i>caf1M</i>(-), and <i>caf1A</i>(-), respectively, indicate that the inner DNA sequences of gene <i>caf1</i>, <i>caf1M</i>, and <i>caf1A</i> were in-frame deleted. (<b>E, F</b>) Comparison of growth rates of P1-pSA, -pSM, -pSF1, and -pY. The bacterial growth rates were determined by measuring the OD₆₀₀ every half hour (<b>E</b>) or determining bacterial CFU every hour (<b>F</b>), and the statistical differences of the growth rates of these strains were calculated using the Tukey Kramer multiple comparisons test. No significant differences of growth rates were discerned among these four strains.</p

Primer sequences and restriction enzyme sites integrated.

<p>Note:</p>a<p>The restriction enzyme site XbaI locates downstream of this primer.</p>b<p>The sequences of the restriction enzyme sites integrated in the primers are bolded.</p>c<p>Primer sequences are based on template of <i>Y. pestis</i> plasmid pFra (accession No. X61996).</p

<i>L</i>. <i>lactis</i>-CFA/I, not <i>L</i>. <i>lactis</i> vector, is a potent therapeutic for collagen-induced arthritis (CIA).

<p>C57BL/6 males (n = 5/group) were CII-challenged on day 0, and treated orally with 5×10⁸ CFUs <i>L</i>. <i>lactis</i>-CFA/I (<i>L</i>. <i>lactis</i>-pBzMM153) or vector control (<i>L</i>. <i>lactis</i>-pMSP3535H3) in sterile PBS or with vehicle alone on days <b>(A)</b> 14, 21, 28 or <b>(B)</b> on days 18 and 25 post-CIA induction as diagramed. Bacteria were grown in synthetic M17 medium supplemented with 0.5% glucose. A representative example of 6 experiments (n = 5/group) is depicted; * <i>p</i> < 0.01; ^✢<i>p</i> < 0.05 as compared to each control group. <b>(C)</b> Joint pathology was evaluated from decalcified knees from mice in each treatment group (n = 5/group). Representative examples of mid-sagittal knee joint sections are stained with H&E (top row) or toluidine blue (bottom row) from mice treated with two doses of <i>L</i>. <i>lactis</i>-CFA/I <b>(B)</b> upon termination of the study.</p