Plasmid-Encoded MCP Is Involved in Virulence, Motility, and Biofilm Formation of Cronobacter sakazakii ATCC 29544

Abstract

bThe aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. Cronobacter spp. are Gram-negative, motile, non-spore-forming, facultative anaerobic microorganisms (1, 2) that have been isolated from a wide range of environments, including water, soil, and a variety of fresh and processed foods, such as powdered milk formula for infants (3-9). The organism is considered to be an opportunistic pathogen and has been linked to life-threatening diseases, including necrotizing enterocolitis, septicemia, and meningitis, with a high mortality rate (40 to 80%) in low-birthweight neonates (7, 9-11). A few reports describe the transmission and virulence of Cronobacter spp.; however, we are still far from completely understanding these mechanisms. Cronobacter spp. can form a biofilm on surfaces, such as glass, stainless steel, polyvinyl chloride, silicone, and enteral feeding tubes, and this biofilm formation could be a vehicle of infection (12)(13)(14). The outer membrane proteins OmpA and OmpX from C. sakazakii are reportedly involved in invasion/adhesion to human enterocyte-like Caco-2 and intestinal INT407 epithelial cells (15)(16)(17). A LysR-type transcriptional regulator (LTTR) reportedly plays a role in various phenotypes that might be important for the transmission and pathogenesis of C. sakazakii, suggesting a role as a global regulator (18).Bacterial plasmids are self-replicating and extrachromosomal replicons that can encode a diverse assortment of virulence factors, including antibiotic resistance, toxins, adherence factors, and secretion systems (19)(20)(21)(22). Plasmid-borne virulence gene clusters of one species have been found in plasmids of other species or pathogenic groups, suggesting acquisition by horizontal gene transfer (20,21). Recently, the genomes of two Cronobacter species, Cronobacter sakazakii ATCC BAA-894 and C. turicensis z3032, have been completely sequenced and shown to po...

Insights into virulence factors determining the pathogenicity ofCronobacter sakazakii

“…96 It was also observed that this sequence was not found in any other lineages, implying that the MCP association with virulence is probably specific to the ST8 lineage.…”

Chemotaxis Control of Transient Cell Aggregation

“…One possibility is that adhesins, which are present in the polar region of the cells or secreted at this location, stabilize the cell-cell contacts in clumps, akin to a similar adhesion described in closely related alphaproteobacteria (59)(60)(61). Flocculation is widespread in many motile bacteria capable of chemotaxis, and chemotaxis defects have been linked to increased cell aggregation in many bacterial species (62)(63)(64). It is thus likely that chemotaxis-dependent clumping occurs in other bacterial species.…”

Draft Genome Sequences of Pandrug-Resistant Serratia marcescens Clinical Isolates Harboring bla NDM-1

“…A notable finding was the presence of a gene with homology to methyl-accepting chemotaxis protein (MCP) on the 4,938 bp-ColRNA1 plasmid. The same gene present on plasmid pCSA2 of Cronobacter sakazakii ATCC29544 was previously shown to be involved in adhesion, invasion, and the regulation of mobility and biofilm formation in that isolate (11). …”