Due to the high incidence of nosocomial Candida albicans infection, the first-line drugs for C. albicans infection have been heavily used, and the emergence of drug-resistant strains has gradually increased. Thus, a new antifungal drug or therapeutic method is needed. Chitosan, a product of chitin deacetylation, is considered to be potentially therapeutic for fungal infections because of its excellent biocompatibility, biodegradability and low toxicity. The biocidal action of chitosan against C. albicans shows great commercial potential, but the exact mechanisms underlying its antimicrobial activity are unclear. To reveal these mechanisms, mutant library screening was performed. ADA2 gene, which encodes a histone acetylation coactivator in the SAGA complex, was identified. Transmission electronic microscopy images showed that the surface of chitosan-treated ada2 Δ cells was substantially disrupted and displayed an irregular morphology. Interestingly, the cell wall of ada2 Δ cells was significantly thinner than that of wild-type cells, with a thickness similar to that seen in the chitosan-treated wild-type strain. Although ADA2 is required for chitosan tolerance, expression of ADA2 and several Ada2-mediated cell wall-related genes ( ALS2, PGA45 , and ACE2 ) and efflux transporter genes ( MDR1 and CDR1 ) were significantly inhibited by chitosan. Furthermore, GCN5 encoding a SAGA complex catalytic subunit was inhibited by chitosan, and gcn5 Δ cells exhibited phenotypes comparable to those of ada2 Δ cells in response to chitosan and other cell surface-disrupting agents. This study demonstrated that a potential antifungal mechanism of chitosan against C. albicans operates by inhibiting SAGA complex gene expression, which decreases the protection of the cell surface against chitosan.
The role of carbapenem-resistant Acinetobacter baumannii (CRAb) in polymicrobial infection remains elusive. Having observed the ability of CRAb to shelter other susceptible bacteria from carbapenem killing, we sought to determine the factors contributing to this sheltering effect by transforming different recombinant plasmids into recipient A. baumannii cells. The sheltering effects of CRAb were reproduced in recipient A. baumannii cells that highly expressed carbapenem-hydrolyzing class D -lactamases (CHDLs) through their associated strong promoter. With the use of Western blot analysis and a bioassay, the highly expressed CHDLs were found to be extracellularly released and led to hydrolysis of carbapenem. The level of extracellular CHDLs increased after challenge with a higher concentration of CHDL substrates, such as carbapenem and ticarcillin. This increased CHDL may, in part, be attributed to cell lysis, as indicated by the presence of extracellular gyrase. In the planktonic condition, the sheltering effect for the cocultured susceptible bacteria might represent an indirect and passive effect of the CRAb self-defense mechanism, because coculture with the susceptible pathogen did not augment the amount of the extracellular CHDLs. Polymicrobial infection caused by CRAb and a susceptible counterpart exerted higher pathogenicity than monomicrobial infection caused by either pathogen alone in mice receiving carbapenem therapy. This study demonstrated that CHDL-producing CRAb appears to provide a sheltering effect for carbapenem-susceptible pathogens via the extracellular release of CHDLs and, by this mechanism, can enhance the pathogenesis of polymicrobial infection in the presence of carbapenem therapy.
f Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D -lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb.A cinetobacter baumannii is a major cause of nosocomial infections worldwide. The rapid emergence of carbapenem-resistant isolates has severely reduced therapeutic options (1, 2). Recently, we demonstrated that carbapenem-resistant A. baumannii (CRAb) sheltered coexisting carbapenem-susceptible bacteria, preventing them from being killed by carbapenem and, thereby, leading to polymicrobial infections with enhanced pathogenicity compared to that of monomicrobial infection (3). This sheltering effect is clinically relevant because 20 to 50% of A. baumannii infections have been found to be polymicrobial (4-6).The primary mechanism of carbapenem resistance in A. baumannii is high-level production of carbapenemases, especially carbapenem-hydrolyzing class D -lactamases (CHDLs), which include the OXA-23, -40, -51, -58, and -143 classes (7). We demonstrated that the extracellular release of CHDLs contributed to the sheltering effect (3), but this was seen only when CHDLs were expressed at high levels using a strong promoter. At the time of the earlier study, the mechanism for the extracellular release of CHDLs had not been elucidated.In this study, we determined that ex...
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