Identification of distinct capsule types associated with Serratia marcescens infection isolates

Abstract

Serratia marcescens is a versatile opportunistic pathogen that can cause a variety of infections, including bacteremia. Our previous work established that the capsule polysaccharide (CPS) biosynthesis and translocation locus contributes to the survival of S. marcescens in a murine model of bacteremia and in human serum. In this study, we determined the degree of capsule genetic diversity among S. marcescens isolates. Capsule loci (KL) were extracted from >300 S. marcescens genome sequences and compared. A phylogenetic comparison of KL sequences demonstrated a substantial level of KL diversity within S. marcescens as a species and a strong delineation between KL sequences originating from infection isolates versus environmental isolates. Strains from five of the identified KL types were selected for further study and electrophoretic analysis of purified CPS indicated the production of distinct glycans. Polysaccharide composition analysis confirmed this observation and identified the constituent monosaccharides for each strain. Two predominant infection-associated clades, designated KL1 and KL2, emerged from the capsule phylogeny. Bacteremia strains from KL1 and KL2 were determined to produce ketodeoxynonulonic acid and N-acetylneuraminic acid, two sialic acids that were not found in strains from other clades. Further investigation of KL1 and KL2 sequences identified two genes, designated neuA and neuB, that were hypothesized to encode sialic acid biosynthesis functions. Disruption of neuB in a KL1 isolate resulted in the loss of sialic acid and CPS production. The absence of sialic acid and CPS production also led to increased susceptibility to internalization by a human monocytic cell line, demonstrating that S. marcescens phagocytosis resistance requires CPS. Together, these results establish the capsule genetic repertoire of S. marcescens and identify infection-associated clades with sialic acid CPS components.

Regulation of Klebsiella pneumoniae mucoidy by the bacterial tyrosine kinase Wzc

“…The pBAD18 backbone, wzc 5' fragment, and mutated wzc 3' fragment were PCR amplified, gel purified (Monarch, NEB), and assembled NEBuilder HiFi DNA Assembly mix (NEB) for 1 h at 60 °C. 47 The ligated products were dialyzed on 0.025 um MCE membranes (MilliporeSigma) against 10% glycerol prior to electroporation. A hexa-histidine tag was added to each pBAD18-Wzc vector with 5'-phosphorylated, non-overlapping primers and T4 ligation.…”

IgaA protein, GumB, has a global impact on the transcriptome and surface proteome of Serratia marcescens

“…Numerous other genes were predicted to be involved in survival were upregulated in the Δ gumB mutant. These include capsule biosynthesis genes that have been associated with pathogenesis (62, 63), uncharacterized genes similar to osmolarity induced genes like osmB, and a predicted metallo-beta-lactamase gene which likely affect stress and antibiotic tolerance.…”

Urine-mediated suppression of Klebsiella pneumoniae mucoidy is counteracted by spontaneous Wzc variants altering capsule chain length

“…Each Wzc point mutation was introduced either by inverse PCR with overlapping mutagenic primers and self-ligation or by inverse PCR with 5′-phosphorylated, non-overlapping primers and T4 ligation. The pBAD18 backbone, wzc 5′ fragment, and mutated wzc 3′ fragment were PCR amplified, gel purified (Monarch, NEB), and assembled using NEBuilder HiFi DNA Assembly mix (NEB) for 1 h at 60°C ( 61 ). The ligated products were dialyzed on 0.025 µm MCE membranes (MilliporeSigma) against 10% glycerol prior to electroporation.…”