Manuel F. Varela

Dexamethasone suppresses pituitary-adrenal but not behavioral effects of centrally administered CRF

Intracerebral ventricular (icv) administration of corticotropin-releasing factor (CRF) significantly enhances the expression of stress-related behaviors in the rat and also activates the pituitary-adrenal system. The pituitary-adrenal response can be blocked by pretreatment of animals with dexamethasone. The behavioral effects (motor activation, increased grooming and decreased eating) on the other hand are resistant to suppression by dexamethasone. The independence of the behavioral effects from activation of the pituitary-adrenal axis suggests that stress-induced release of CRF could contribute to behavioral alterations even in the presence of high concentrations of endogenous steroids.

LmrS Is a Multidrug Efflux Pump of the Major Facilitator Superfamily from Staphylococcus aureus

ABSTRACT A multidrug efflux pump designated LmrS (lincomycin resistance protein of Staphylococcus aureus), belonging to the major facilitator superfamily (MFS) of transporters, was cloned, and the role of LmrS in antimicrobial efflux was evaluated. The highest relative increase in MIC, 16-fold, was observed for linezolid and tetraphenylphosphonium chloride (TPCL), followed by an 8-fold increase for sodium dodecyl sulfate (SDS), trimethoprim, and chloramphenicol. LmrS has 14 predicted membrane-spanning domains and is homologous to putative lincomycin resistance proteins of Bacillus spp., Lactobacillus spp., and Listeria spp.

Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens

Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations.

Biochemistry of Bacterial Multidrug Efflux Pumps

Bacterial pathogens that are multi-drug resistant compromise the effectiveness of treatment when they are the causative agents of infectious disease. These multi-drug resistance mechanisms allow bacteria to survive in the presence of clinically useful antimicrobial agents, thus reducing the efficacy of chemotherapy towards infectious disease. Importantly, active multi-drug efflux is a major mechanism for bacterial pathogen drug resistance. Therefore, because of their overwhelming presence in bacterial pathogens, these active multi-drug efflux mechanisms remain a major area of intense study, so that ultimately measures may be discovered to inhibit these active multi-drug efflux pumps.

Modulation of Bacterial Multidrug Resistance Efflux Pumps of the Major Facilitator Superfamily

Bacterial infections pose a serious public health concern, especially when an infectious disease has a multidrug resistant causative agent. Such multidrug resistant bacteria can compromise the clinical utility of major chemotherapeutic antimicrobial agents. Drug and multidrug resistant bacteria harbor several distinct molecular mechanisms for resistance. Bacterial antimicrobial agent efflux pumps represent a major mechanism of clinical resistance. The major facilitator superfamily (MFS) is one of the largest groups of solute transporters to date and includes a significant number of bacterial drug and multidrug efflux pumps. We review recent work on the modulation of multidrug efflux pumps, paying special attention to those transporters belonging primarily to the MFS.

SugE, a New Member of the SMR Family of Transporters, Contributes to Antimicrobial Resistance in Enterobacter cloacae

ABSTRACT We cloned a gene, sugE, from the chromosome of Enterobacter cloacae ATCC 13047. Analysis of the susceptibilities of the sugE-containing strain (Escherichia coli KAM32/pSUGE28) and sugE-deficient E. cloacae (EcΔsugE) showed that SugE confers resistance to cetyltrimethylammonium bromide, cetylpyridinium chloride, tetraphenylphosphonium, benzalkonium chloride, ethidium bromide, and sodium dodecyl sulfate. We also investigated expression of sugE. We confirm here that SugE from E. cloacae is an SMR family transporter as determined by observing its energy-dependent drug efflux activity.

Physiological evidence for an interaction between Glu-325 and His-322 in the lactose carrier of Escherichia coli

Site-directed mutagenesis and second-site suppressor analysis have proven to be useful approaches to examine the role of charged amino acids in the structure and function of the lactose carrier of Escherichia coli. A lactose carrier mutant Glu-325 --> Ser failed to ferment melibiose and showed white clones on melibiose MacConkey indicator plates. Several red revertants were isolated from these plates. Two of these revertants showed a double mutation, the original mutation (Glu-325 --> Ser) plus His-322 --> Asp. Seven revertants showed a second site mutation His-322 --> Asn. Although the second site revertants failed to accumulate sugars they do show more rapid uptake of melibiose into cells containing alpha-galactosidase than the original mutant Glu-325 --> Ser. The complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a new negative charge at 322. A site-directed double mutant His-322 --> Asn/Glu-325 --> Asn showed a greater rate of lactose uptake (Vmax) than either of the single mutants His-322 --> Asn or Glu 325 --> Asn. It was concluded that there is some type of physiological interaction (possibly a salt bridge) between His-322 and Glu-325.

Nucleotide and deduced protein sequences of the class D tetracycline resistance determinant: relationship to other antimicrobial transport proteins.

The nucleotide sequence of the plasmid pRA1 gene encoding the TetA(D) tetracycline/H+ antiporter was determined. The deduced amino acid sequence was compared with those of other antimicrobial and antiseptic transporters. The deduced product of tetA(D) is a 41.1-kDa protein consisting of 394 amino acids comprising 12 membrane-spanning domains. Three classes of amino acid motifs found in TetA(D) are highly conserved in other transporters, implying that they participate in structures necessary for substrate recognition, binding, or translocation. A common mechanism of transport is suggested, with subtle sequence variations accounting for varied substrate specificities, modes of transport, and directions of transport.

Age-related changes in the motor response to environmental novelty in the rat

To examine age-related changes in responsiveness to environmental novelty, 3-, 12-, and 24-month-old male Fischer 344 rats were maintained on a restricted diet and exposed to a modified open field for 10 min on each of 10 consecutive days. On the first day of testing, animals of all groups showed equal amounts of rearing. While the 3-month animals continued to show approximately the same levels of rearing until the 8th day, by day 5, the older groups (12- and 24-month) had significantly reduced their rearing. Conversely, grooming was initially highest among the 24-month-old animals. While 3- and 12-month rats showed habituation of grooming, the oldest animals failed to habituate their grooming response by day 5. By day 10, there were no significant differences among the groups in either rearing or grooming. Although food was available in the center of the modified open field, there was little eating and there were no differences among groups. However, all animals did eat quickly when food was made available in their home cages. Thus, all animals displayed a profile of stress-related responses to open field exposure. Plasma corticosterone levels likewise were elevated in the modified open field. Some, but not all, components of this response profile habituated over the 10 days of exposure. Three-month-old animals responded to the novelty predominantly by rearing and 24-month animals predominantly by grooming. This suggests that with aging, locomotor responses to stress are replaced by a more self-directed form of displacement activity.

EmmdR, a new member of the MATE family of multidrug transporters, extrudes quinolones from Enterobacter cloacae

We cloned a gene, ECL_03329, from the chromosome of Enterobacter cloacae ATCC13047, using a drug-hypersensitive Escherichia coli KAM32 cell as the host. We show here that this gene, designated as emmdR, is responsible for multidrug resistance in E. cloacae.E. coli KAM32 host cells containing the cloned emmdR gene (KAM32/pEMMDR28) showed decreased susceptibilities to benzalkonium chloride, norfloxacin, ciprofloxacin, levofloxacin, ethidium bromide, acriflavine, rhodamine6G, and trimethoprim. emmdR-deficient E. cloacae cells (EcΔemmdR) showed increased susceptibilities to several of the antimicrobial agents tested. EmmdR has twelve predicted transmembrane segments and some shared identity with members of the multidrug and toxic compound extrusion (MATE) family of transporters. Study of the antimicrobial agent efflux activities revealed that EmmdR is an H+-drug antiporter but not a Na+ driven efflux pump. These results indicate that EmmdR is responsible for multidrug resistance and pumps out quinolones from E. cloacae.