Flow cytometric analysis of CCL28 binding to the surface of wild type and mutant bacteria.

<p>In chemokine binding assays bacteria were incubated with CCL28 followed by biotinylated anti-chemokine antibody and APC-streptavidin conjugates. Negative controls included the omission of chemokine followed by the incubation of bacteria with anti-chemokine antibody followed by streptavidin-APC. Results indicate that wild-type <i>Y</i>. <i>pseudotuberculosis</i> is not readily bound by CCL28 (A). Conversely, transposon mutant insertions in the <i>manC</i> (B) or <i>hldD</i> (C) genes greatly increase the number of bacteria bound by chemokines.</p

LPS analysis.

<p>The <i>hldD</i>::Tn5 mutant shows no O- antigen expression and the presence of a truncated core oligosaccharide. Addition of <i>hldD</i>, <i>hldD/waaF</i>, or <i>hldD/waaF/waaC</i> plasmids cause production of full-length core oligosaccharide and O- antigen to increase. As expected, <i>Y</i>. <i>pseudotuberculosis</i> grown at 37°C does not show O- antigen expression. Sample IDs (from left to right) 1—wild type <i>Y</i>. <i>pseudotuberculosis</i> IP 32953 grown at 21°C, 2—<i>hldD</i>::Tn5 mutant, 3 -<i>hldD</i>+, 4 –<i>hldD/waaF</i>+, 5—<i>hldD/waaF/waaC</i> +, 6—wild type <i>Y</i>. <i>pseudotuberculosis</i> grown at 37°C. LPS was detected by fluorescent staining of carbohydrates.</p

Role of the <i>hldD/waaF/waaC</i> operon in survival against CCL28.

<p>Relative survival of bacteria (expressed as a percentage of the number of live cells counted in the unexposed control of the same strain) in the presence of CCL28 over a 5 h exposure. Asterisks denote strains that were significantly different from the wild type strain at that time point (Two-way ANOVA (****p < 0.0001).</p

Independent Tn5 insertion mutants in LPS-related genes with increased AMC binding phenotypes.

<p>Independent Tn5 insertion mutants in LPS-related genes with increased AMC binding phenotypes.</p

Role of HldD, WaaF, and WaaC in polymyxin resistance.

<p>Relative survival of bacteria (expressed as a percentage of the number of live cells counted in the unexposed control of the same strain) in the presence of different concentrations of polymyxin B. Asterisks denote that the result obtained was significantly different from the wild type strain at the given concentration by Two-way ANOVA (****p < 0.0001, ***p < 0.001, **p< 0.01, *p < 0.05).</p

Role of HldD, WaaF, WaaC in AMC binding.

<p>(A) A mutation in <i>hldD</i> results in significantly greater CCL25 and CCL28 binding (p<0.0001 by Two-way ANOVA), expressed as a percentage of cells that stain positive as measured by flow cytometry. In the <i>hldD</i>::Tn5 mutant background, addition of <i>hldD/waaF</i> slightly reduced CCL28 (**p<0.01) and CCL25 (*p<0.05) binding, whereas the presence of all three genes (<i>hldD/waaF/waaC</i>) completely reduced binding affinity for both CCL25 and CCL28 to wild-type levels (****p<0.0001) when grown at 21°C. (B) Enhanced CCL28 binding due to <i>hldD</i> mutation is also evident in bacteria expressing low levels of O-antigen from cultures grown at 37°C.</p

Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria

<div><p>In addition to the well characterized function of chemokines in mediating the homing and accumulation of leukocytes to tissues, some chemokines also exhibit potent antimicrobial activity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. Here we describe the cloning and function of bovine CCL28 and document the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.</p></div

Primer names and sequences.

<p>Primer names and sequences.</p

Bovine CCL28 mediates migration of CCR10 transfected cells Supernatant fluids from Cos-7 cells transfected with mCCL28, bCCL28, or empty vector controls were placed in the bottom well of a Transwell migration chamber.

<p>Cells expressing mCCR10 were placed in the upper chamber and allowed to migrate for 1.5hrs. Both murine and bovine CCL28 mediated migration of CCR10 transfectants significantly better than empty vector controls *p<0.05, as determined by Mann Whitney <i>U</i> test. Results are from four separate experiments. Average migration of cells migrating to mCCL28 was 18.4% (SE 3.71), migration to bCCL28 9.4% (SE 2.12), and migration to empty vector 0.1% (SE 0.01). Error bars represent standard error of the mean. NS = Not Significant.</p

Recombinant bCCL28 has potent broad spectrum antimicrobial activity against mastitis causing bacteria.

<p>Bovine CCL28 demonstrates dose dependent antimicrobial activity against <i>Pseudomonas aeruginosa</i> (<i>A)</i>, methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) <i>(B)</i>, <i>Streptococcus uberis (C)</i>, and <i>Streptococcus agalactiae (D)</i>. Bacteria were incubated with varied doses of bCCL28 or bCCL27 for 1 hour. Percent survival was calculated using the following equation: (CFU_{pathogen + chemokine}/CFU_{pathogen only}) x 100. *p<0.05, **p<0.01 as determined by Mann Whitney <i>U</i> test. Results are from four or more separate experiments. Error bars represent standard error of the mean. Details on percent survival, standard error, and the number of times each experiment was performed are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138084#pone.0138084.s001" target="_blank">S1 Fig</a>.</p