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

Abstract

Avidocin-CDs are a new class of precision bactericidal agents that do not damage resident gut microbiota and are unlikely to promote the spread of antibiotic resistance. The precision killing properties result from the fusion of bacteriophage receptor binding proteins (RBPs) to a lethal contractile scaffold from an R-type bacteriocin. We recently described the prototypic Avidocin-CD, Av-CD291.2, that specifically kills C. difficile ribotype 027 strains and prevents colonization of mice. We have since selected two rare Av-CD291.2 resistant mutants of strain R20291 (RT027; S-layer cassette type-4, SCLT-4). These mutants have distinct point mutations in the slpA gene that result in an S-layer null phenotype. Reversion of the mutations to wild-type restored normal SLCT-4 S-layer formation and Av-CD291.2 sensitivity; however, complementation with other SCLT alleles did not restore Av-CD291.2 sensitivity despite restoring S-layer formation. Using newly identified phage RBPs, we constructed a panel of new Avidocin-CDs that kill C. difficile isolates in an SLCT-dependent manner, confirming the S-layer as the receptor in every case. In addition to bacteriophage adsorption, characterization of the S-layer null mutant also uncovered important roles for SlpA in sporulation, resistance to lysozyme and LL-37, and toxin production. Surprisingly, the S-layer-null mutant was found to persist in the hamster gut despite its completely attenuated virulence. Avidocin-CDs have significant therapeutic potential for the treatment and prevention of C. difficile Infection (CDI) given their exquisite specificity for the pathogen. Furthermore, the emergence of resistance forces mutants to trade virulence for continued viability and, therefore, greatly reduce their potential clinical impact.

Cwp22, a novel peptidoglycan cross‐linking enzyme, plays pleiotropic roles in Clostridioides difficile

“…The high-molecular-weight SLP (about 47 kDa) acts as a binding subunit, and the low-molecular-weight SLP (about 36 kDa) modulates colonization and pathogenesis (Takeoka et al, 1991;Calabi et al, 2001). The role of the C. difficile S-layer in colonization, immunity, virulence and viability has been well-studied (Calabi et al, 2002;Pechine et al, 2005;Ausiello et al, 2006;Pechine et al, 2007;Kirk et al, 2017). Several other CWPs such as Cwp84, Cwp2, Cwp66, and CwpV have been investigated, and play important roles in host cell adhesion and immune system evasion during CDI (Waligora et al, 2001;Emerson et al, 2009;Kirby et al, 2009;Bradshaw et al, 2017;Wydau-Dematteis et al, 2018).…”

Spatial organization of Clostridium difficile S-layer biogenesis

“…The 197 distribution of signal in these images suggest that SlpA secretion occurs over the majority of 198 the cell's surface and not just at sites where new S-layer is being formed. 199 200 Discussion 201For an S-layer to function correctly it must completely encapsulate the cell (de la Riva,Willing, 202 Tate, & Fairweather, 2011;Kirk et al, 2017). We propose here that S-layer is assembled at 203 areas of newly synthesized peptidoglycan to maintain a stable S-layer that continually protects204 the C. difficile cell.…”

“…During exponential growth, C. difficile cells are constantly growing and dividing, requiring the 63 production of new peptidoglycan at the cell wall. The S-layer protects the cell envelope from 64 innate immune effectors such as lysozyme and LL-37 (Kirk et al, 2017). This function requires 65 that an S-layer barrier is maintained while new peptidoglycan is synthesized during growth.…”

“…Clostridium difficile infection (CDI) is the major cause of antibiotic associated diarrhoea (Hull 24 & Beck, 2004) and can lead to severe inflammatory complications (Napolitano & Edmiston, 25 2017). This Gram-positive bacterium has a cell wall encapsulating, proteinaceous surface-layer 26 (S-layer), a paracrystalline array that acts as a protective semipermeable shell and is essential 27 for virulence (Kirk et al, 2017). In C. difficile the S-layer consists mainly of SlpA, the most 28 abundant surface protein (Calabi et al, 2001).…”

“…DNA modifications were performed using Phusion High-262 Fidelity DNA Polymerase (Thermo Fisher) and Q5 Site-Directed Mutagenesis Kit (NEB) as 263 per manufacturers' instructions. 264Plasmid Construction 265pRFP233(Kirk et al, 2017) was modified to add the tetracysteine (Tc) tag encoding sequence 266 into slpA such that a modified LMW-SLP is produced with FLNCCPGCCMEP added to a 267 surface-exposed loop. The plasmid was linearized by inverse PCR using oligonucleotides 268 RF411 and RF412, deleting 150 bp of the LMW-SLP coding sequence.…”