A <i>Clostridium difficile</i> Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence

<div><p><i>Clostridium difficile</i> is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in <i>C</i>. <i>difficile</i> pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five <i>C</i>. <i>difficile</i> CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in <i>C</i>. <i>difficile</i> infection is unknown. We explored the contribution of a conserved CWG locus to <i>C</i>. <i>difficile</i> cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter <i>mviN</i>, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated <i>mviN</i> downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, <i>lcpA</i> and <i>lcpB</i>, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. <i>lcpA</i>^- and <i>lcpB</i>^- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The <i>lcpB</i>^- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both <i>lcpA</i>^- and <i>lcpB</i>^- strains also displayed heightened virulence in a hamster model of <i>C</i>. <i>difficile</i> disease. We propose that gene products of the <i>C</i>. <i>difficile</i> CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence.</p></div

Strains, plasmids and primers used in this study.

<p>Strains, plasmids and primers used in this study.</p

<i>lcpB</i> disruption remodels the bacterial cell surface.

<p>(A) Surface-layer Protein (SLP) profiling of <i>C</i>. <i>difficile</i> strains. Comparable SLP profiles of the isogenic parent [WT (Vector)], <i>lcpA</i>^- mutant [<i>lcpA</i>^- (vector)] and complement (<i>lcpA</i>^- p<i>lcpA</i>; Lanes 1, 2 and 3). Altered SLP profile of the <i>lcpB</i>^- mutant [<i>lcpB</i>^- (vector); Lane 4] revealing additional low molecular weight products. Complementation-based restoration of the <i>lcpB</i>^- mutant SLP profile (<i>lcpB</i>^- p<i>lcpB</i>; Lane 5). Three independent biological replicates were performed for each strain; a representative image is shown. (B) Biofilm formation by <i>C</i>. <i>difficile</i> strains. The <i>lcpA</i>^- mutant and complement produce comparable biofilms to the parent strain (Bars 1, 2 and 3), but the <i>lcpB</i>^- mutant [<i>lcpB</i>^- (vector)] produces a more robust biofilm, that is only partially restored to wild-type levels via plasmid complementation (Bars 4 and 5). Three biological replicates (each in technical triplicate) were performed for each strain. The error bars are represented by standard deviation estimates as well as Student’s <i>t</i> tests to compute differences between WT (Vector) and <i>lcpB</i>^- (Vector). Significance is <i>p</i> < 0.05.</p

<i>C</i>. <i>difficile</i> encodes a putative CWG locus.

<p>(A) The orientation of the genes in the putative CWG locus is shown along with the generalized predicted functions of each gene product [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005946#ppat.1005946.ref021" target="_blank">21</a>]. The color of each gene corresponds to its predicted function shown in the legend below the locus. (B) The locus is highly conserved at the amino acid level (95%-100%) across diverse <i>C</i>. <i>difficile</i> strains. A total of 32 clinical isolates were analyzed and compared to the reference strain CD630 (boxed in green). A list of all the strains used is provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005946#ppat.1005946.s001" target="_blank">S1 Table</a>.</p

<i>C</i>. <i>difficile lcpB</i> disruption profoundly impacts bacterial morphology.

<p>Columns 1 and 2: Scanning electron micrographs of <i>C</i>. <i>difficile</i> strains in low resolution (1000X; Column 1, scale bar is 10μm), and high resolution (10,000X, Column 2, scale bar is 2.5μm). Curved, elongated morphotype of the <i>lcpB</i>^- mutant [<i>lcpB</i>^- (vector)] is shown in Row 2, and complementation-based rescue in Row 3. There was no obvious phenotype for the <i>lcpA</i>^- strain. Column 3: Transmission electron micrographs (TEM) of parent strain (Row 1), <i>lcpB</i>^- mutant [Row 2; <i>lcpB</i>^- (vector)]) and complement (Row 3). Improper septum formation (yellow arrow) and a diffuse cell wall structure (black arrow) is shown. Scale bar for TEM is 100nm. All images are representative of a minimum of 10 fields visualized, and at least two biological replicate preparations.</p

<i>C</i>. <i>difficile lcpB</i> impacts PSII production and localization.

<p>(A) Immunoblot analysis demonstrating increased shedding of PSII from the <i>lcpB</i>^- strain [<i>lcpB</i>^- (Vector); Row 4] compared to the isogenic parent [WT (Vector); Row 1]. Shed PSII levels are restored in a plasmid-complemented strain (<i>lcpB</i>^- p<i>lcpB</i>, Row 5). Minimal increase in shed PSII from the <i>lcpA</i>^- mutant [<i>lcpA</i>^- (Vector); Row 2] and corresponding plasmid complementation (<i>lcpA</i>^- p<i>lcpA</i>; Row 3) is also shown. UD, “undiluted”. All immunoblots are representative of a minimum of two biological replicates. (B) Immunofluorescence demonstrating PSII co-staining with the cell-surface-specific dye FM4-64 on the isogenic parent (Row 1 images). Alteration and re-localization of PSII in the <i>lcpA</i>^- strain (Row 2 images) and <i>lcpB</i>^- strain (Row 4 images). Altered morphology, with a curved and elongated phenotype and multiple septae are also visible in the <i>lcpB</i>^- strain. Complementation-mediated restoration of PSII co-localization with FM4-64 in both mutants is shown in Rows 3 and 5 images respectively, as well as the morphology defect rescue in <i>lcpB</i>^- strain (Row 5 images).</p

<i>C</i>. <i>difficile</i> PSII biosynthesis model [22,23,48].

<p>One repeating unit of PSII is assembled on a lipid carrier (undecaprenyl phosphate) in the bacterial cytoplasm, exported to the cell surface, polymerized and anchored to peptidoglycan, the cell membrane, or cell wall proteins. Sequentially: (1) a predicted initiating transferase (CD2783) transfers the first sugar of the PSII repeating unit to the lipid carrier; (2) a second glycosyltransferase catalyzes the committed step of the pathway by transferring the second sugar to the repeating unit, and cytoplasmic glycosyltransferases, including ManC and Pgm2, synthesize the rest of the PSII unit; (3) a polysaccharide flippase (MviN) transports the PSII repeating unit from the cytoplasm to the cell surface; (4) a polymerase (CD2777) polymerizes the PSII chain extracellularly and the lipid carrier is recycled by an unknown mechanism (indicated by the asterisk); (5) finally, surface-anchoring factors (LcpA and/or LcpB) catalyze the transfer and anchoring of fully polymerized PSII to peptidoglycan or the cell membrane. Consistent with the NMR determinations derived in Ganeshapillai et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005946#ppat.1005946.ref015" target="_blank">15</a>], we have included the phosphate unit only on the mannose residue on polymerized PSII. However, the mechanistic basis of this phospho-sugar linkage has not been explored.</p

<i>lcp</i> disruption impacts <i>C</i>. <i>difficile</i> virulence.

<p>(A) Pilot study. The blue bar represents one uninfected hamster, the red bar is one wild-type infected hamster, the purple bars are <i>lcpA</i>^- infected hamsters (4 total) and the green bars are <i>lcpB</i>^- infected hamsters (5 total). (B) Confirmation of <i>lcpB</i>^- strain hypervirulence in a powered study. A Kaplan-Meier survival plot is shown; n = 5 for parent strain-infected animals, and n = 10 for <i>lcpB</i>^- mutant infected animals. Bacteria with helical morphology are visualized in the cecal contents of <i>lcpB</i>^- mutant infected hamsters (inset).</p

<i>C</i>. <i>difficile mviN</i> impacts cell-surface moieties.

<p>Decreased <i>mviN</i> expression in GV341 (strain expressing <i>mviN</i> anti-sense RNA: KD, “knock-down”) compared to GV342 or GV349 (vector control strains) results in less extractable PSII based on immunodot-blot analysis (A) and less PSII on the cell surface visualized by immunofluorescence microscopy (B). The ramp in (A) indicates increasing to decreasing concentration from left to right, UD is “undiluted,” and the scale bar in (B) represents 10μm.</p