The Prevalence of Exoenzyme S Gene in Multidrug-Sensitive and Multidrug-Resistant Pseudomonas aeruginosa Clinical Strains

Pseudomonas aeruginosa rods are one of the most commonly isolated microorganisms from clinical specimens, usually responsible for nosocomial infections. Antibiotic-resistant P. aeruginosa strains may present reduced expression of virulence factors. This fact may be caused by appropriate genome management to adapt to changing conditions of the hospital environment. Virulence factors genes may be replaced by those crucial to survive, like antimicrobial resistance genes. The aim of this study was to evaluate, using PCR, the occurrence of exoenzyme S-coding gene (exoS) in two distinct groups of P. aeruginosa strains: 83 multidrug-sensitive (MDS) and 65 multidrug-resistant (MDR) isolates. ExoS gene was noted in 72 (48.7%) of the examined strains: 44 (53.0%) MDS and 28 (43.1%) MDR. The observed differences were not statistically significant (p = 0.1505). P. aeruginosa strains virulence is rather determined by the expression regulation of the possessed genes than the difference in genes frequency amongst strains with different antimicrobial susceptibility patterns.

Exoenzyme S ADP-Ribosylates Rab5 Effector Sites To Uncouple Intracellular Trafficking

ABSTRACTPseudomonas aeruginosaexoenzyme S (ExoS) ADP-ribosylates multiple eukaryotic targets to promote cytopathology and bacterial colonization. ADP-ribosylation of the small GTPase Rab5 has previously been shown to block fluid-phase endocytosis and trafficking of plasma membrane receptors to the early endosomes as well as inhibit phagocytosis of the bacterium. In this study, ExoS is shown to be capable of ADP-ribosylating 6 candidate arginine residues that are located in the effector binding region or in the C terminus of Rab5. Two Rab5 derivatives were engineered, which contained Arg→Ala mutations at four Arg residues within the effector binding region (EF) or two Arg residues within the C-terminal tail (TL). Expression of Rab5(TL) does not affect the ability of ExoS to modify intracellular trafficking, while expression of Rab5(EF) rescued the ability of ExoS to inhibit intracellular trafficking. ADP-ribosylation of effector arginines likely uncouples Rab5 signaling to downstream effectors. This is a different mechanism for inhibition than observed for the ADP-ribosylation of Ras by ExoS, where ADP-ribosylated Ras loses the ability to bind guanine nucleotide exchange factor (GEF). Other experiments showed that expression of dominant negative Rab5(Ser34Asn) does not inhibit ExoS trafficking to the perinuclear region of intoxicated cells. This study provides insight into a mechanism for how ExoS ADP-ribosylation of Rab5 inhibits Rab5 function.

SELECTIVE USE OF MEMBRANE CD14, BUT NOT SOLUBLE CD14 ANDINHIBITION BY SERUM IN THE INFLAMMATORY CYTOKINE RESPONSE INDUCED BY A NOVELTLR LIGAND (P. AERUGINOSA EXOENZYME S)

Background/Purpose: Soluble andmembrane CD14 (sCD14 and mCD14) bind microbial products, are toll-like receptor(TLR) coreceptors, and induce inflammation. Clinical relevance is demonstrated by a correlation of sCD14 levels with morbidity and mortality in inflammatory diseases. Moreover, P. aeruginosa induces damaging inflammation in the lungs of cysticfibrosis patients and its virulence is often attributed to the TLR2 and TLR4ligand exoenzyme S (ExoS). Because of the importance of the response of CD14 toTLR ligands and its potential for therapeutic manipulation, its role inExoS-induced inflammatory cytokines was investigated. Methods: The ability of ExoS to induce TNFand IL-6 cytokine production in cell lines that express TLR and either CD14 positive or negative was assessed (THP-1 or U373 cells, respectively). Recombinant CD14 or CD14 blocking agents were used to test the contribution ofmCD14 and sCD14. Results: Enhancing expression of mCD14 on THP-1 cells increased TNF production, which was abrogated by blocking or removing mCD14. Transfecting mCD14 into U373 cells demonstrated that mCD14 was required for binding ExoS and subsequent IL-6 production. Unlike TLR ligands that only stimulate one TLR, such as lipopolysaccharide, neither sCD14 nor serum enhanced production of cytokine in response to ExoS. Uniquely, serum inhibited ExoS induced TNF. Conclusion: This work demonstrates a fundamental difference in the requirement of ExoS and other TLR ligands for CD14, which must be considered when designing therapies to block microbe-induced inflammation. The presence of a potential therapeutic molecule in serum could help in the development of an ExoS neutralizing agent.