A global view of antibiotic resistance.

Antibiotic resistance is one of the few examples of evolution that can be addressed experimentally. The present review analyses this resistance, focusing on the networks that regulate its acquisition and its effect on bacterial physiology. It is widely accepted that antibiotics and antibiotic resistance genes play fundamental ecological roles - as weapons and shields, respectively - in shaping the structures of microbial communities. Although this Darwinian view of the role of antibiotics is still valid, recent work indicates that antibiotics and resistance mechanisms may play other ecological roles and strongly influence bacterial physiology. The expression of antibiotic resistance determinants must therefore be tightly regulated and their activity forms part of global metabolic networks. In addition, certain bacterial modes of life can trigger transient phenotypic antibiotic resistance under some circumstances. Understanding resistance thus requires the analysis of the regulatory networks controlling bacterial evolvability, the physiological webs affected and the metabolic rewiring it incurs.

The global regulator Crc modulates metabolism, susceptibility to antibiotics and virulence in Pseudomonas aeruginosa.

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross-talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high-throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing-regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta-lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.

Proteomics of RAW 264.7 macrophages upon interaction with heat-inactivated Candida albicans cells unravel an anti-inflammatory response.

Murine macrophages (RAW 264.7) were allowed to interact with heat-inactivated cells of Candida albicans SC5314 during 45 min. The proteomic response of the macrophages was then analyzed using 2-D gel electrophoresis. Many proteins having differential expression with respect to control macrophages were identified, and their functions were related to important processes, such as cytoskeletal organization, signal transduction, metabolism, protein biosynthesis, stress response and protein fate. Several of these proteins have been described as being involved in the process of inflammation, such as Erp29, Hspa9a, AnxaI, Ran GTPase, P4hb, Clic1 and Psma1. The analysis of the consequences of their variation unravels an overall anti-inflammatory response of macrophages during the interaction with heat-inactivated cells. This result was corroborated by the measurement of TNF-alpha and of ERK1/2 phosphorylation levels. This anti-inflammatory effect was contrary to the one observed with live C. albicans cells, which induced higher TNF-alpha secretion and higher ERK1/2 phosphorylation levels with respect to control macrophages.

Chronic Pseudomonas aeruginosa infection in chronic obstructive pulmonary disease.

BACKGROUND: Pseudomonas aeruginosa infections are increasingly associated with acute exacerbations in chronic obstructive pulmonary disease (COPD). We aimed to determine whether an underlying chronic infection might be behind this process and to determine the epidemiological characteristics of the isolates involved, to implement useful protocols for preventing and treating these infections. METHODS: P. aeruginosa isolates obtained from respiratory samples of 13 patients with COPD and from blood samples of 10 patients in intensive care units were investigated. In 8 patients with COPD, isolates were obtained during sequential exacerbation episodes. Five patients presented a single infection episode. Production of virulence determinants and genetic relationships were analyzed in all isolates. RESULTS: Patients with COPD were usually infected with 1 P. aeruginosa clone that remained in the lung for years, without evidence of interpatient transmission. During chronic infection, each clone diversified, which led to the coexistence of isolates with different morphotypes and antibiotic susceptibility. Overall, P. aeruginosa evolved toward an increased mutation rate, increased antibiotic resistance, and reduced production of proteases. Isolates from samples of infected lungs tend to be less cytotoxic and motile and to produce more biofilm, compared with isolates from blood samples. CONCLUSION: These results provide the first evidence supporting the hypothesis that P. aeruginosa causes chronic infections in COPD, with patterns of infection and evolution that resemble those observed in cystic fibrosis. Experience gained from treating cystic fibrosis might be useful for implementing new procedures for the prevention, diagnosis, and treatment of infection due to P. aeruginosa in COPD.

Differential protein expression of murine macrophages upon interaction with Candida albicans.

Numerous studies highlight the importance of macrophages for optimal host protection against systemic Candida albicans infections. We chose the murine macrophage cell line RAW 264.7 and the wild-type strain C. albicans SC5314 to study of the induced expression/repression of proteins in macrophages when they are in contact with C. albicans, based on 2-DE, comparison between different gels and protein identification. RAW 264.7 cells were allowed to interact with C. albicans cells for 45 min, and a significant differential protein expression was observed in these macrophages compared to controls. Gels were stained with SYPRO Ruby, allowing a better quantification of the intensity of the protein spots. Fifteen spots were up-regulated, whereas 32 were down-regulated; 60 spots appeared and 49 disappeared. Among them, we identified 11 proteins: annexin I, LyGDI (GDID4), Hspa5 (Grp78, Bip), tropomyosin 5 and L-plastin, that augment; and Eif3s5, Hsp60, Hspa9a, Grp58 (ER75), and Hspa8a (Hsc70), that decrease. The translation elongation factor (Eef2p) is modified in some of its different protein species. Many processes seem to be affected: cytoskeletal organisation, oxidative responses (superoxide and nitric oxide production) and protein biosynthesis and refolding.

Sub-proteomic study on macrophage response to Candida albicans unravels new proteins involved in the host defense against the fungus.

In previous proteomic studies on the response of murine macrophages against Candida albicans, many differentially expressed proteins involved in processes like inflammation, cytoskeletal rearrangement, stress response and metabolism were identified. In order to look for proteins important for the macrophage response, but in a lower concentration in the cell, 3 sub-cellular extracts were analyzed: cytosol, organelle/membrane and nucleus enriched fractions from RAW 264.7 macrophages exposed or not to C. albicans SC5314 for 3 h. The samples were studied using DIGE technology, and 17 new differentially expressed proteins were identified. This sub-cellular fractionation permitted the identification of 2 mitochondrion proteins, a membrane receptor, Galectin-3, and some ER related proteins, that are not easily detected in total cell extracts. Besides, the study of different fractions allowed us to detect, not only total increase in Galectin-3 protein amount, but its distinct allocation along the interaction. The identified proteins are involved in the pro-inflammatory and oxidative responses, immune response, unfolded protein response and apoptosis. Some of these processes increase the host response and others could be the effect of C. albicans resistance to phagocytosis. Thus, the sub-proteomic approach has been a very useful tool to identify new proteins involved in macrophage-fungus interaction. This article is part of a Special Issue entitled: Translational Proteomics.

Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems.

Multidrug efflux pumps have emerged as relevant elements in the intrinsic and acquired antibiotic resistance of bacterial pathogens. In contrast with other antibiotic resistance genes that have been obtained by virulent bacteria through horizontal gene transfer, genes coding for multidrug efflux pumps are present in the chromosomes of all living organisms. In addition, these genes are highly conserved (all members of the same species contain the same efflux pumps) and their expression is tightly regulated. Together, these characteristics suggest that the main function of these systems is not resisting the antibiotics used in therapy and that they should have other roles relevant to the behavior of bacteria in their natural ecosystems. Among the potential roles, it has been demonstrated that efflux pumps are important for processes of detoxification of intracellular metabolites, bacterial virulence in both animal and plant hosts, cell homeostasis and intercellular signal trafficking.