Role of the Multidrug Efflux Systems of Pseudomonas aeruginosa in Organic Solvent Tolerance

Multidrug efflux pumps with a broad substrate specificity make a major contribution to intrinsic and acquired multiple antibiotic resistance in Pseudomonas aeruginosa. Using genetically defined efflux pump mutants, we investigated the involvement of the three known efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, in organic solvent tolerance in this organism. Our results showed that all three systems are capable of providing some level of tolerance to organic solvents such as n-hexane and p-xylene. Expression of MexAB-OprM was correlated with the highest levels of tolerance, and indeed, this efflux system was a major contributor to the intrinsic solvent tolerance of P. aeruginosa. Intrinsic organic solvent tolerance was compromised by a protonophore, indicating that it is substantially energy dependent. These data suggest that the efflux of organic solvents is a factor in the tolerance of P. aeruginosa to these compounds and that the multidrug efflux systems of this organism can accommodate organic solvents, as well as antibiotics.     

Influence of the MexAB-OprM Multidrug Efflux System on Quorum Sensing in Pseudomonas aeruginosa

Pseudomonas aeruginosa nalB mutants which hyperexpress the MexAB-OprM multidrug efflux system produce reduced levels of several extracellular virulence factors known to be regulated by quorum sensing. Such mutants also produce less acylated homoserine lactone autoinducer PAI-1, consistent with an observed reduction in lasI expression. These data suggest that PAI-1 is a substrate for MexAB-OprM, and its resulting exclusion from cells hyperexpressing MexAB-OprM limits PAI-1-dependent activation of lasI and the virulence genes.     

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.     

茶多酚对金黄色葡萄球菌和铜绿假单胞菌的抑菌机理

以革兰氏阳性的金黄色葡萄球菌和革兰氏阴性的铜绿假单胞菌为试验菌,通过测定茶多酚与两种菌作用前后细菌培养液的电导率和可溶性总糖的变化,以及菌体在磷代谢和蛋白质表达方面的变化,初步阐明了茶多酚对这两种菌的抑菌机理。研究结果表明,茶多酚对金黄色葡萄球菌和铜绿假单胞菌均有抑菌活性,但对金黄色葡萄球菌的抑菌活性更强。经茶多酚处理后,细菌培养液的电导率和总糖浓度均增大,表明了茶多酚可破坏细胞膜的结构、导致细胞通透性增加,进而使细胞内容物外泄。另一方面,经茶多酚处理后的两种菌对磷的消耗量降低,以致严重影响了核酸、磷脂等细胞重要成分的合成以及能量代谢;通过SDS-PAGE分析,证实茶多酚可以阻碍细菌蛋白质的正常表达,以致影响其细胞的结构组成以及酶的催化活性,最终导致细菌正常生理功能的丧失。     

肠杆菌科AcrAB多药外排泵分子演化分析

目的：分析肠杆菌科AcrAB多药外排泵的分子演化规律,为多药耐药性病原菌的防治提供基础数据。方法：从NCBI获得肠杆菌科物种AcrAB多药外排泵相关蛋白和核酸序列,采用分析软件,分析肠杆菌科物种AcrAB多药外排泵相关序列。结果：在肠杆菌科各物种AcrA、AcrB和AcrR与大肠埃希氏菌同源性在55%、75%和43%以上。AcrA保守位点分散,在N端和C端较少,在分子一级结构中段较多。AcrB跨膜序列保守性较高,与质子转移相关的三个位点D407、D408和K940以及稳定这三者结构的T978在肠杆菌科完全保守,其一级结构上相邻位点也保守。AcrR序列整体保守性较低,但HTH区域保守性高。与AcrR结合的回文结构及周围序列保守性高,在＂茎＂结构中仅存在一个氨基酸的差异。结论：AcrAB多药外排泵在肠杆菌科中广泛存在,有一定的保守性。分析肠杆菌科AcrAB多药外排泵有助于病原菌多药耐药性的防治。     

Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na⁺- or Li⁺-dependent manner. Moreover, Na⁺ efflux via PdrM was observed when acriflavine was added to Na⁺-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na⁺/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.     

Antimicrobial resistance of Pseudomonas aeruginosa biofilms

Resistance to antimicrobial agents is the most important feature of biofilm infections. As a result, infections caused by bacterial biofilms are persistent and very difficult to eradicate. Although several mechanisms have been postulated to explain reduced susceptibility to antimicrobials in bacterial biofilms, it is becoming evident that biofilm resistance is multifactorial. The contribution of each of the different mechanisms involved in biofilm resistance is now beginning to emerge.     

Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India

ObjectivesThe present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India.Methods76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed.ResultsOut of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35^th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump.ConclusionsThis study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options.     

Multidrug Efflux Pump MdtBC of Escherichia coli Is Active Only as a B2C Heterotrimer

RND (resistance-nodulation-division) family transporters in Gram-negative bacteria frequently pump out a wide range of inhibitors and often contribute to multidrug resistance to antibiotics and biocides. An archetypal RND pump of Escherichia coli, AcrB, is known to exist as a homotrimer, and this construction is essential for drug pumping through the functionally rotating mechanism. MdtBC, however, appears different because two pump genes coexist within a single operon, and genetic deletion data suggest that both pumps must be expressed in order for the drug efflux to occur. We have expressed the corresponding genes, with one of them in a His-tagged form. Copurification of MdtB and MdtC under these conditions showed that they form a complex, with an average stoichiometry of 2:1. Unequivocal evidence that only the trimer containing two B protomers and one C protomer is active was obtained by expressing all possible combinations of B and C in covalently linked forms. Finally, conversion into alanine of the residues, known to form a proton translocation pathway in AcrB, inactivated transport only when made in MdtB, not when made in MdtC, a result suggesting that MdtC plays a different role not directly involved in drug binding and extrusion.Bacterial multidrug resistance is a major public health problem (10, 17). One widespread resistance mechanism involves the multidrug resistance (MDR) transporters. Among these, the resistance-nodulation-cell division (RND) family transporters, such as the AcrAB-TolC system in Escherichia coli, play a major role in drug resistance in Gram-negative bacteria because they allow the direct extrusion of drug molecules into extracellular space, and because they sometimes confer an increased level of tolerance to an astonishingly wide range of toxic compounds (18). In general, an RND-type exporter protein (such as AcrB), located in the inner membrane, forms a tripartite complex with a periplasmic adaptor protein, such as AcrA, and a homotrimeric outer membrane channel, such as TolC (18). The drug efflux process requires the presence of all three components. The crystallographic structures of AcrB (13, 14, 22, 24), AcrA (11, 27), and TolC (2, 8) are known, and models of the tripartite complex have been proposed (6, 27).AcrB is a homotrimeric transporter (14) located in the inner membrane and uses the proton gradient as the energy source (31). The homotrimeric structure is thought to be functionally important, or even essential, as each protomer appears to undergo a series of mandatory conformational alterations during the process of drug export, often called “functionally rotating mechanism,” as deduced from the structure of the asymmetric trimers of AcrB (13, 22, 24). This mechanism was also supported by the observation that, in a trimer in which protomers were covalently linked to each other, inactivation of one protomer alone abolishes the activity of the entire trimeric complex (29).Not all RND-type transporters, however, follow this homotrimeric organization. The mdtABC genes of E. coli encode an RND system that is unusual in that it contains two different RND pump genes, mdtB and mdtC, in addition to its own adaptor gene, mdtA. Previous genetic studies have demonstrated that the deletion of either of the two RND pump genes abolishes (1) the resistance to β-lactams, novobiocin, and bile salt derivatives, like deoxycholate, or narrows the range of pump substrates (15), a result suggesting that the functional unit is likely a heteromultimeric pump formed by MdtB/MdtC proteins. However, no direct data have so far been presented supporting the interaction between these proteins or the stoichiometry of the complex. Because the heterooligomeric composition of this pump was unexpected based on the accepted notion of how the homotrimeric pump functions by the functionally rotating mechanism, we examined here the nature of the MdtBC complex in detail.In this study, we first purified the oligomeric transporter by labeling either MdtB or MdtC with a His tag. We obtained a trimeric complex(es) containing both MdtB and MdtC in an approximately 2:1 ratio. However, we could not rule out the possibility that there were mixtures of trimers containing different ratios of the B and C proteins. We therefore utilized the recently introduced technology of creating covalently linked trimers (29), and we show here that the only active trimers are those containing two units of MdtB and one unit of MdtC.     

Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Suggests an Alternative Assembly Mode

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.     

Action-mode of Antimicrobial Altersolanol A in Pseudomonas aeruginosa

The antimicrobial mechanism of altersolanol A was studied by using Pseudomonas aeruginosa IFO 3080. This reduced anthraquinone antibiotic inhibited the incorporation of radioactive precursors into macromolecules (DNA, RNA, and Protein) in whole cells nonspecifically. It increased respiration of P. aeruginosa cells, though it had no effect on proton condution. Oxidation of NADH in the membrane fraction isolated from P. aeruginosa was enhanced by altersolanol A. Furthermore, altersolanol A stimulated the oxidation of NADH by the enzyme preparation of cytochrome c reductase in the absence of cytochrome c. Altersolanol A seemed to interfere with the respiratory chain in the bacterial membrane as an electron acceptor.     

Study on Antiviral Activity of Two Recombinant Antimicrobial Peptides Against Tobacco Mosaic Virus

Probiotics and Antimicrobial Proteins - Antimicrobial peptides (AMPs) are generally small peptides with less than 50 amino acid residues, which have been considered as the first line of defense...     

Antimicrobial activities of Saudi honey against Pseudomonas aeruginosa

Five types of imported and local honey were screened for both their bacteriocidal/bacteriostatic activities against both Imipenem resistant and sensitive Pseudomonas aeruginosa in both Brain Heart infusion broth and Mueller–Hinton agar. The results indicated that the effect was concentration and type of honey dependant. All types of honey tested exerted a full inhibition of bacterial growth at the highest concentration tested of 50% at 24 h of contact. The inhibitory effect of honey on bacterial growth was clear with concentrations of 20% and 10% and this effect was most evident in the case of Manuka honey as compared to Nigella sativa honey and Seder honey. Manuka honey UMF +20 showed a bacteriocidal activity on both Imipenem resistant and sensitive P. aeruginosa, while Seder honey and N. sativa honey exerted only a bacteriostatic effect. Manuka honey UMF +10 showed most effect on antimicrobial resistance. Manuka honey UMF +10 had an effect on modulation of Imipenem resistant P. aeruginosa. Conclusion: The results indicated that various types of honey affected the test organisms differently. Modulation of antimicrobial resistance was seen in the case Manuka honey UMF +10.     

Human Antimicrobial Peptides: Defensins, Cathelicidins and Histatins

Antimicrobial peptides, which have been isolated from many bacteria, fungi, plants, invertebrates and vertebrates, are an important component of the natural defenses of most living organisms. The isolated peptides are very heterogeneous in length, sequence and structure, but most of them are small, cationic and amphipathic. These peptides exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts, fungi and enveloped viruses. A wide variety of human proteins and peptides also have antimicrobial activity and play important roles in innate immunity. In this review we discuss three important groups of human antimicrobial peptides. The defensins are cationic non-glycosylated peptides containing six cysteine residues that form three intramolecular disulfide bridges, resulting in a triple-stranded β-sheet structure. In humans, two classes of defensins can be found: α-defensins and β-defensins. The defensin-related HE2 isoforms will also be discussed. The second group is the family of histatins, which are small, cationic, histidine-rich peptides present in human saliva. Histatins adopt a random coil conformation in aqueous solvents and form α-helices in non-aqueous solvents. The third group comprises only one antimicrobial peptide, the cathelicidin LL−37. This peptide is derived proteolytically from the C-terminal end of the human CAP18 protein. Just like the histatins, it adopts a largely random coil conformation in a hydrophilic environment, and forms an α-helical structure in a hydrophobic environment.