Elucidating the Regulon of the Multidrug Resistance Regulator RarA in Klebsiella pneumoniae

RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also been shown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8?rarA/pACrarA-2 (rarA-expressing construct) and Ecl8?rarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found to be downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype of K. pneumoniae.

Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system

Colistin resistance is rare in Acinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient. pmrAB sequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression of pmrA (4- to 13-fold), pmrB (2- to 7-fold), and pmrC (1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg induction conditions. pmrB gene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression of pmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance in A. baumannii requires distinct genetic events, including (i) at least one point mutation in pmrB, (ii) upregulation of pmrAB, and (iii) expression of pmrC, which lead to addition of phosphoethanolamine to lipid A. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.

Identification of the lipopolysaccharide core of Yersinia pestis and Yersinia pseudotuberculosis as the receptor for bacteriophage φA1122

fA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. fA1122 infects Y. pestis grown both at 20 °C and at 37 °C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37 °C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30 °C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also fA1122 sensitive when grown at 22 °C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous fA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (L-glycero-D-manno-heptose/D-glucopyranose)-Kdo/Ko (3-deoxy-D-manno-oct-2-ulopyranosonic acid/D-glycero-D-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully fA1122 resistant. Our data thus conclusively demonstrated that the fA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.

Analysis of the networks controlling the antimicrobial-peptide-dependent induction of Klebsiella pneumoniae virulence factors

Antimicrobial peptides (APs) impose a threat to the survival of pathogens, and it is reasonable to postulate that bacteria have developed strategies to counteract them. Polymyxins are becoming the last resort to treat infections caused by multidrug-resistant Gram-negative bacteria and, similar to APs, they interact with the anionic lipopolysaccharide. Given that polymyxins and APs share the initial target, it is possible that bacterial defense mechanisms against polymyxins will be also effective against host APs. We sought to determine whether exposure to polymyxin will increase Klebsiella pneumoniae resistance to host APs. Indeed, exposure of K. pneumoniae to polymyxin induces cross-resistance not only to polymyxin itself but also to APs present in the airways. Polymyxin treatment upregulates the expression of the capsule polysaccharide operon and the loci required to modify the lipid A with aminoarabinose and palmitate with a concomitant increase in capsule and lipid A species containing such modifications. Moreover, these surface changes contribute to APs resistance and also to polymyxin-induced cross-resistance to APs. Bacterial loads of lipid A mutants in trachea and lungs of intranasally infected mice were lower than those of wild-type strain. PhoPQ, PmrAB, and the Rcs system govern polymyxin-induced transcriptional changes, and there is a cross talk between PhoPQ and the Rcs system. Our findings support the notion that Klebsiella activates a defense program against APs that is controlled by three signaling systems. Therapeutic strategies directed to prevent the activation of this program could be a new approach worth exploring to facilitate the clearance of the pathogen from the airways.

Role of lipid A acylation in Yersinia enterocolitica virulence

Yersinia enterocolitica is an important human pathogen. Y. enterocolitica must adapt to the host environment, and temperature is an important cue regulating the expression of most Yersinia virulence factors. Here, we report that Y. enterocolitica 8081 serotype O:8 synthesized tetra-acylated lipid A at 37 degrees C but that hexa-acylated lipid A predominated at 21 degrees C. By mass spectrometry and genetic methods, we have shown that the Y. enterocolitica msbB, htrB, and lpxP homologues encode the acyltransferases responsible for the addition of C(12), C(14) and C(16:1), respectively, to lipid A. The expression levels of the acyltransferases were temperature regulated. Levels of expression of msbB and lpxP were higher at 21 degrees C than at 37 degrees C, whereas the level of expression of htrB was higher at 37 degrees C. At 21 degrees C, an lpxP mutant was the strain most susceptible to polymyxin B, whereas at 37 degrees C, an htrB mutant was the most susceptible. We present evidence that the lipid A acylation status affects the expression of Yersinia virulence factors. Thus, expression of flhDC, the flagellar master regulatory operon, was downregulated in msbB and lpxP mutants, with a concomitant decrease in motility. Expression of the phospholipase yplA was also downregulated in both mutants. inv expression was downregulated in msbB and htrB mutants, and consistent with this finding, invasion of HeLa cells was diminished. However, the expression of rovA, the positive regulator of inv, was not affected in the mutants. The levels of pYV-encoded virulence factors Yops and YadA in the acyltransferase mutants were not affected. Finally, we show that only the htrB mutant was attenuated in vivo.

Klebsiella pneumoniae AcrAB efflux pump contributes to antimicrobial resistance and virulence

Respiratory infections caused by Klebsiella pneumoniae are characterized by high rates of mortality and morbidity. Management of these infections is often difficult, due to the high frequency of strains that are resistant to multiple antimicrobial agents. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens. In the present study, we investigated the role of the K. pneumoniae AcrRAB operon in antimicrobial resistance and virulence by using isogenic knockouts deficient in the AcrB component and the AcrR repressor, both derived from the virulent strain 52145R. We demonstrated that the AcrB knockout was more susceptible, not only to quinolones, but also to other antimicrobial agents, including beta-lactams, than the wild-type strain and the AcrR knockout. We further showed that the AcrB knockout was more susceptible to antimicrobial agents present in human bronchoalveolar lavage fluid and to human antimicrobial peptides than the wild-type strain and the AcrR knockout. Finally, the AcrB knockout exhibited a reduced capacity to cause pneumonia in a murine model, in contrast to the wild-type strain. The results of this study suggest that, in addition to contributing to the multidrug resistance phenotype, the AcrAB efflux pump may represent a novel virulence factor required for K. pneumoniae to resist innate immune defense mechanisms of the lung, thus facilitating the onset of pneumonia.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.

Klebsiella pneumoniae increases the levels of Toll-like receptors 2 and 4 in human airway epithelial cells

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IkappaBalpha-dependent NF-kappaBeta pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.

Klebsiella pneumoniae OmpA confers resistance to antimicrobial peptides

A Klebsiella pneumoniae ompA mutant was more susceptible to antimicrobial peptides (APs) than the wild type. Susceptibility did not result from surface changes other than the absence of OmpA. Our data suggest that OmpA is implicated in the activation of yet-unknown systems dedicated to ameliorating AP cytotoxicity.

Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.

Expression of the Yersinia enterocolitica pYV-encoded type III secretion system is modulated by lipopolysaccharide O-antigen status

We show that the expression of a Yersinia enterocolitica O:8 pYV-encoded type III secretion system was altered in a rough mutant (YeO8-R) due to elevated levels of FlhDC. H-NS might underlie flhDC upregulation in YeO8-R, and the data suggest a relationship between the absence of O antigen and the expression of H-NS.

Quinolones sensitize gram-negative bacteria to antimicrobial peptides

The treatment of infections caused by bacteria resistant to the vast majority of antibiotics is a challenge worldwide. Antimicrobial peptides (APs) make up the front line of defense in those areas exposed to microorganisms, and there is intensive research to explore their use as new antibacterial agents. On the other hand, it is known that subinhibitory concentrations of antibiotics affect the expression of numerous bacterial traits. In this work we evaluated whether treatment of bacteria with subinhibitory concentrations of quinolones may alter the sensitivity to APs. A 1-h treatment of Klebsiella pneumoniae with 0.25 x the MIC of ciprofloxacin rendered bacteria more sensitive to polymyxins B and E, human neutrophil defensin 1, and beta-defensin 1. Levofloxacin and nalidixic acid at 0.25 x the MICs also increased the sensitivity of K. pneumoniae to polymyxin B, whereas gentamicin and ceftazidime at 0.25 x the MICs did not have such an effect. Ciprofloxacin also increased the sensitivities of K. pneumoniae ciprofloxacin-resistant strains to polymyxin B. Two other pathogens, Pseudomonas aeruginosa and Haemophilus influenzae, also became more sensitive to polymyxins B and E after treatment with 0.25 x the MIC of ciprofloxacin. Incubation with ciprofloxacin did not alter the expression of the K. pneumoniae loci involved in resistance to APs. A 1-N-phenyl-naphthylamine assay showed that ciprofloxacin and levofloxacin increased the permeabilities of the K. pneumoniae and P. aeruginosa outer membranes, while divalent cations antagonized this action. Finally, we demonstrated that ciprofloxacin and levofloxacin increased the binding of APs to the outer membrane by using dansylated polymyxin B.

Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.