A Mig-14-like protein (PA5003) affects antimicrobial peptide recognition in Pseudomonas aeruginosa

Abstract

The evolution of antibiotic resistance in pathogenic bacteria is a growing global health problem which is gradually making the treatment of infectious diseases less efficient. Antimicrobial peptides are small charged molecules found in organisms from the complete phylogenetic spectrum. The peptides are attractive candidates for novel drug development due to their activity against bacteria that are resistant to conventional antibiotics, and reports of peptide resistance are rare in the clinical setting. Paradoxically, many clinically relevant bacteria have mechanisms that can recognize and respond to the presence of cationic antimicrobial peptides (CAMPs) in the environment by changing the properties of the microbial surface thereby increasing the tolerance of the microbes towards the peptides. In Pseudomonas aeruginosa an essential component of this inducible tolerance mechanism is the lipopolysaccharide modification operon arnBCADTEF-PA3559 which encodes enzymes required for LPS alterations leading to increased antimicrobial peptide tolerance. The expression of the operon is induced by the presence of CAMPs in the environment but the molecular mechanisms underlying the cellular recognition of the peptides are poorly elucidated. In this work, we investigate the factors influencing arnB expression by transposon mutagenesis and arnB promoter green fluorescent protein reporters. We have identified a novel gene encoding a Mig-14-like protein that is required for recognition of the CAMPs colistin and Novispirin G10 by P. aeruginosa. Moreover, we show that this gene is also required for the formation of CAMP-tolerant subpopulations in P. aeruginosa hydrodynamic flow chamber biofilms. INTRODUCTIONAntimicrobial peptides are an integral part of the host defence against infection and have been found in most multi-cellular organisms (Zasloff, 2002). The peptides are small, often cationic and structurally diverse and display broad-spectrum antimicrobial activity against a range of bacteria (Brogden, 2005). Charge interaction between the cationic peptide and the anionic microbial membrane is thought to be a critical step in a process leading to microbial killing (Brogden, 2005). A number of other peptide activities have been identified, indicating roles as multifunctional mediators of immunity, inflammation and wound repair (Hiemstra et al., 2004). Cationic antimicrobial peptides (CAMPs) are evident alternatives for drug development due to their activity on bacteria resistant to conventional antibiotics (Hiemstra et al., 2004). The polymyxins such as colistin are cyclic CAMPs which are highly active against many Gram-negative bacteria and are extensively used in the treatment of Pseudomonas aeruginosa lung infections in patients with cystic fibrosis (Frederiksen et al., 1997;Hoeprich, 1970;Littlewood et al., 1985;Storm et al., 1977). The appearance of bacteria resistant to most classes of antibiotics and the shortage of novel antimicrobial agents with activity against Gramnegative micro-organisms have led to the reemergence ...

Host Defense Peptide Resistance Contributes to Colonization and Maximal Intestinal Pathology by Crohn's Disease-Associated Adherent-Invasive Escherichia coli

“…1A) (38)(39)(40). One of these genes (NRG857_30278 or arlA) is an ortholog to a gene involved in antimicrobial peptide resistance in Salmonella (38) and Pseudomonas aeruginosa (41). The second gene (NRG857_30283 or arlC) is a probable paralog of an ompT family member protease that has emerged as a more widely distributed antimicrobial peptide protease (42,43).…”

“…The mechanism by which Mig-14 provides resistance to host defense peptides is not fully known. Proteins from this class are associated with the inner membrane where they are thought to decrease the penetrance of active peptide to the inner cytoplasmic membrane (39,41). Mig-14 family members seem to mediate resistance to a wide range of structural classes of peptides, including colistin, novaspirin G10, CRAMP, and protegrin, which is why ArlA might have the greatest impact on in vivo fitness.…”

Determinants of Intrinsic Aminoglycoside Resistance in Pseudomonas aeruginosa

“…As such, these two systems may regulate overlapping stress responses such that the full impact of a stress-response defect vis-à-vis aminoglycoside susceptibility is not felt until both are absent. While the probable sigma factor controlled by PA2797-PA2798 is unknown, disruption of this locus has recently been linked to increased colistin tolerance (45), although a mechanism was not elucidated.…”

Polymyxin Resistance of Pseudomonas aeruginosa phoQ Mutants Is Dependent on Additional Two-Component Regulatory Systems

“…Aside from phoPQ itself (PA1179-1180), only two individual genes were implicated in both studies: galU (PA2023) and mpl (PA4020). Both of these studies also implicated adjacent genes (wapR-ssg-hypothetical-mig14-wapH, PA5000-5004) within the LPS core biosynthesis gene cluster (PA4996-5012) (41,42). Among the other 12 genes implicated in Mg 2ϩ depletion-induced PMB resistance (70), pmrA (PA4776) and parR (PA1798) were described previously (16,19,26,27).…”

“…The transposon analysis implicated cell wall biogenesis genes, such as oprF (PA1777), galU (PA2023), oprI (PA2853), prc (PA3257), and mpl (PA4020); the pagL gene (PA4661) encoding a lipid A deacylase (39,40); genes within the LPS core biosynthesis cluster (PA5002-5004), including wapH and mig-14 (41,42); and algC (PA5322), a gene required for biosynthesis of full-length LPS (41). The analysis also implicated a locus (PA4476) encoding a large hypothetical protein with C-terminal homology to the AsmA proteins of E. coli and Yersinia pestis; null mutations in asmA are associated with decreased LPS levels in E. coli (43), while induction of asmA transcription is associated with increased antimicrobial peptide resistance in Y. pestis (44).…”