Abstract
Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells.
Highlights
Enterobacter cloacae are Gram-negative, facultative anaerobic and rod-shaped bacteria belonging to the order Enterobacterales (McAdam, 2020)
type 6 secretion system (T6SS)-1 appears to be complete as it contains hcp secreted protein-encoding gene as well as the clpV ATPase-encoding gene, among other core and accessory gene components (Figure 1; Supplementary Table S1), suggesting that T6SS-1 could be functional in E. cloacae
We evaluated gene expression of both T6SS-1 and T6SS2 on 6 h of growth of E. cloacae in different culture media by RT-qPCR, determining the mRNA levels of three different genes of each genetic cluster, which are the first genes of putative operons
Summary
Enterobacter cloacae are Gram-negative, facultative anaerobic and rod-shaped bacteria belonging to the order Enterobacterales (McAdam, 2020). These bacteria are saprophytic in the environment and are part of the human gut microbiota (Mezzatesta et al, 2012). E. cloacae is a human opportunistic pathogen that is frequently associated with hospital-acquired infections of the lower respiratory tract, urinary tract, and meninges (Liu et al, 2013). Despite the relevance of E. cloacae as a nosocomial pathogen, its pathogenicity mechanisms are not yet fully understood, but biofilm formation is a virulence. Two T6SS Are Required for E. cloacae Virulence feature of this opportunistic microorganism (Zhou et al, 2014; Zurob et al, 2019). Some E. cloacae strains possess cytotoxic activity, suggesting the secretion of bacterial toxins to the cell host (Barnes et al, 1997; Krzyminska et al, 2009)
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