The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile

Abstract

The dlt operon in Gram-positive bacteria encodes proteins that are necessary for the addition of D-alanine to teichoic acids of the cell wall. The addition of D-alanine to the cell wall results in a net positive charge on the bacterial cell surface and, as a consequence, can decrease the effectiveness of antimicrobials, such as cationic antimicrobial peptides (CAMPs). Although the roles of the dlt genes have been studied for some Gram-positive organisms, the arrangement of these genes in Clostridium difficile and the life cycle of the bacterium in the host are markedly different from those of other pathogens. In the current work, we determined the contribution of the putative C. difficile dlt operon to CAMP resistance. Our data indicate that the dlt operon is necessary for full resistance of C. difficile to nisin, gallidermin, polymyxin B and vancomycin. We propose that the D-alanylation of teichoic acids provides protection against antimicrobial peptides that may be essential for growth of C. difficile in the host. INTRODUCTIONClostridium difficile causes a potentially fatal intestinal disease that is increasing in incidence and severity (Dubberke et al., 2010). Infection is often chronic and difficult to eradicate (O'Brien et al., 2007). C. difficile is transmitted as dormant endospores that are highly resistant to killing by heat, antibiotics, oxidizing agents, UV radiation and many other treatments that are usually effective for killing bacteria (Vonberg et al., 2008;Setlow, 1995). C. difficile spores germinate in the intestine to produce vegetatively growing cells capable of producing toxins A and B, which have been implicated in disease progression and severity (Bartlett et al., 1977;McDonald et al., 2005).Upon germination, actively growing C. difficile must contend with the onslaught of host defences present in the intestinal environment. The host produces a number of innate immune factors, such as cationic antimicrobial peptides (CAMPs) and bacteriolytic enzymes that protect the intestine from invading bacterial pathogens (Müller et al., 2005;Gudmundsson & Agerberth, 1999). Some of these CAMPs are products of host cells and others are produced by the intestinal microflora. These host effectors are attracted to the overall negative charge of bacterial cells and, as a consequence, bacterial resistance to these effectors can be achieved by charge modification of cell wall and membrane components (Nizet, 2006;Peschel, 2002). In many Gram-positive bacteria, this charge modification occurs by D-alanyl esterification of teichoic acid in the cell wall, which results in an increased positive charge on the cell (Neuhaus & Baddiley, 2003). The addition of D-alanine esters to teichoic acids is typically mediated by the products of the dlt operon, which encodes four proteins: DltA, a D-alanine : D-alanyl carrier protein ligase; DltB, a D-alanyl transfer protein; DltC, the D-alanyl carrier protein; and DltD, a D-alanine esterase. All four proteins are required for successful addition of D-alanine to the cell wall (Ne...

Efficacy of Lantibiotic Treatment of Staphylococcus aureus-Induced Skin Infections, Monitored byIn VivoBioluminescent Imaging

“…However, resistance to lantibiotics has been described in the literature, and the majority of the mechanisms responsible for resistance involve alterations in the charge and permeability of the cell wall or membrane, respectively (37). Resistance mechanisms include alteration of the cell wall and membrane, such as increases in the positive charge of the cell wall or changes in the phospholipid composition of the cell membrane (37)(38)(39)(40)(41)(42)(43)(44)(45)(46). Other resistance mechanisms include biofilms, spore formation, and, in some cases, specific antilantibiotic mechanisms (37).…”

Lantibiotic Resistance

“…Hence, cationic antimicrobial peptides (CAMPs), such as nisin and gallidermin, are repelled from the cell envelope of target microorganisms, such as L. monocytogenes, Bacillus cereus, Clostridium difficile, Streptococcus pneumoniae, and Staphylococcus aureus (45,(47)(48)(49)(50). This is a form of innate lantibiotic resistance and becomes particularly evident in the presence of antimicrobial peptides that trigger a signaling pathway that upregulates the process (45).…”

“…In Streptococcus agalactiae, two regulatory genes of the dlt operon, designated dltR and dltS, are located upstream of dltA (46). In Clostridium difficile, a putative regulatory protein (CD2850) is thought to negatively regulate the dlt operon (47).…”

Regulation of bacterial virulence gene expression by cell envelope stress responses

“…Consistent with this, dlt null mutants of Gram-positive pathogens have increased sensitivity to CAMPs, increased killing by human neutrophils and reduced virulence in animal models of infection. [156][157][158][159][160][161][162][163][164] The dlt operon is also one of the best examples of virulence genes controlled by ESRs in Gram-positive bacteria. Even though many Gram-positive ESRs are widely conserved, their target genes can vary significantly between species.…”