New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability

Abstract

There is a medical need for antibacterial agents that do not damage the resident gut microbiota or promote the spread of antibiotic resistance. We recently described a prototypic precision bactericidal agent, Av-CD291.2, which selectively kills specific Clostridium difficile strains and prevents them from colonizing mice. We have since selected two Av-CD291.2-resistant mutants that have a surface (S)-layer-null phenotype due to distinct point mutations in the slpA gene. Using newly identified bacteriophage receptor binding proteins for targeting, we constructed a panel of Avidocin-CDs that kills diverse C. difficile isolates in an S-layer sequence-dependent manner. In addition to bacteriophage receptor recognition, characterization of the mutants also uncovered important roles for S-layer protein A (SlpA) in sporulation, resistance to innate immunity effectors, and toxin production. Surprisingly, S-layer-null mutants were found to persist in the hamster gut despite a complete attenuation of virulence. These findings suggest antimicrobials targeting virulence factors dispensable for fitness in the host force pathogens to trade virulence for viability and would have clear clinical advantages should resistance emerge. Given their exquisite specificity for the pathogen, Avidocin-CDs have substantial therapeutic potential for the treatment and prevention of C. difficile infections.

Fig. 1

Mutations in slpA confer Av-CD291.2 resistance. (A) Alignment of the slpA sequence (nucleotides 268-294) from R20291, FM2.5, FM2.6, FM2.5RW and FM2.6RW. A nucleotide insertion at position 283 of FM2.5 slpA results in a frameshift and premature stop codon (in blue). A nucleotide substitution at position 280 of FM2.6 slpA results in a nonsense mutation (in red). To allow differentiation from the wild type sequence, two synonymous mutations were introduced into slpA in FM2.5RW and FM2.6RW (in green) (B) SDS-PAGE analysis of S-layer extracts from R20291, FM2.5, FM2.6, FM2.5RW and FM2.6RW. The positions of the LMW and HMW SLPs and minor cell wall proteins Cwp2 and Cwp6 are indicated. (C and D) The impact of Av-CD291.2 on exponentially growing R20291, FM2.5, FM2.5RW and FM2.6RW was monitored by measuring the optical density at 600 nm. Av-D291.2 addition is indicated with an arrow. Experiments were carried out in triplicate on biological duplicates. Means and standard deviations are shown. (E) SDS-PAGE analysis and Av-CD291.2 sensitivity of FM2.5 complemented with slpA alleles from multiple SLCTs following induction with anhydrotetracycline (20 ng/ml). R20291 and FM2.5RW are included as controls. A zone of clearance in the agar lawn indicates killing.

Fig. 2

Avidocin-CD sensitivity correlates with SLCT. (A) SDS-PAGE analysis of SLPs extracted from a panel of strains representing the 11 most commonly isolated SLCTs. (B) Spot bioassays with 8 Avidocin-CDs on the C. difficile strains used in panel A, as well as FM2.5 alone (-) and FM2.5 complemented with slpA alleles from 10 SLCTs following induction with anhydrotetracycline (20 ng/ml). The zone of clearance caused by each Avidocin-CD is shown along with SLCT (H = Hybrid 2/6).

Fig. 3

Phenotypic characterization of FM2.5. (A and B) Cultures of R20291, FM2.5 and FM2.5RW were challenged with lysozyme (500 μg/ml; A) or LL-37 (5 μg/ml; B) in exponential phase after 2.5 h (indicated with arrows). Untreated control cultures were grown in parallel. Experiments were carried out in triplicate on biological duplicates. Means and standard deviations are shown. (C) Sporulation of R20291, FM2.5, and FM2.5RW after 5 days. Spore CFUs were determined following a standard 65°C heat treatment for 30 minutes or a harsher 75°C heat treatment for 30 minutes. Heat-resistant spore CFUs are expressed as a percentage of total viable CFUs (spores and vegetative cells). Experiments were carried out in duplicate on biological duplicates. Mean and standard deviation are shown. * = P<0.01, determined using using two-tailed t-tests with Welch's correction. (D) Germination of R20291, FM2.5, and FM2.5RW spores. Synchronous germination of purified spores was induced with the bile salt taurocholate. Germination initiation was monitored by measuring the resulting decrease in optical density at OD_600nm.

Fig. 4

In vivo analysis of slpA mutant in the Syrian Golden hamster. (A) Times to experimental endpoint of animals infected with R20291 (black line), FM2.5 (dark blue line) and FM2.5RW (light blue line) respectively. Each line represents 6 animals. (B) Total CFUs and spore CFUs (following heat treatment at 56°C for 20 min) were determined for Lumen- (LA) and tissue-associated (TA) bacteria recovered from caecum (CAE) and colon (COL) of infected animals and quantified at experimental endpoint (R20291 and FM2.5RW) or at 14 days post-infection (FM2.5). Shown are the mean and standard error. The horizontal dotted line indicates the limit of detection. None of the observed differences, including those for the TA-spore CFUs from the colon, are statistically significant. (C and D) Relative toxin activity of filtered gut samples on HT-29 (toxin A) and Vero cells (toxin B) respectively. Values represent the reciprocal of the first dilution in which cell morphology was indistinguishable from untreated wells. Samples were taken at experimental endpoint (R20291 and FM2.5RW) or at 14 days post-infection (FM2.5). (* = P <0.05, ** = P <0.01, NS = not significant, determined using using a two-tailed nonparametric Mann-Whitney test. (E) In vitro cell lysate and culture supernatant samples from R20291, FM2.5 and FM2.5RW were normalized to an equivalent optical density and separated on 6% SDS polyacrylamide gels. Toxin B was detected by Western immunoblot using an anti-Toxin B monoclonal antibody. Samples were taken at the indicated time points.

Fig. 5

Schematic diagram depicting the phenotypes of Wild Type (A) and S-layer null mutant (B) cells in C. difficile biology and pathophysiology. Processes labeled in black indicate new functions discerned in this manuscript. Processes labeled in grey indicate previously identified functions. Dotted lines indicate relationships that need to be studied further. Question marks indicate previously described connections incompatible with observations made with the S-layer null mutant and warranting further investigation. (C) Schematic of Avidocin-CD structure and function. Binding to C. difficile by the Avidocin-CD receptor binding proteins (RBPs) triggers the sheath to contract and force the hollow nanotube core across the cell envelope. The resulting pore allows small metabolites, such as protons, ATP and cations, to escape from the cell cytoplasm, which, in turn, disrupts the cell’s membrane potential and kills the cell.