Hansi Kumari

A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa is a metabolically versatile bacterium that is found in a wide range of biotic and abiotic habitats. It is a major human opportunistic pathogen causing numerous acute and chronic infections. The critical traits contributing to the pathogenic potential of P. aeruginosa are the production of a myriad of virulence factors, formation of biofilms and antibiotic resistance. Expression of these traits is under stringent regulation, and it responds to largely unidentified environmental signals. This review is focused on providing a global picture of virulence gene regulation in P. aeruginosa. In addition to key regulatory pathways that control the transition from acute to chronic infection phenotypes, some regulators have been identified that modulate multiple virulence mechanisms. Despite of a propensity for chaotic behaviour, no chaotic motifs were readily observed in the P. aeruginosa virulence regulatory network. Having a ‘birds-eye’ view of the regulatory cascades provides the forum opportunities to pose questions, formulate hypotheses and evaluate theories in elucidating P. aeruginosa pathogenesis. Understanding the mechanisms involved in making P. aeruginosa a successful pathogen is essential in helping devise control strategies.

Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response

Pathogenicity of Pseudomonas aeruginosa, a major cause of many acute and chronic human infections, is determined by tightly regulated expression of multiple virulence factors. Quorum sensing (QS) controls expression of many of these pathogenic determinants. Previous microarray studies have shown that the AmpC β-lactamase regulator AmpR, a member of the LysR family of transcription factors, also controls non-β-lactam resistance and multiple virulence mechanisms. Using RNA-Seq and complementary assays, this study further expands the AmpR regulon to include diverse processes such as oxidative stress, heat shock and iron uptake. Importantly, AmpR affects many of these phenotypes, in part, by regulating expression of non-coding RNAs such as rgP32, asRgsA, asPrrF1 and rgRsmZ. AmpR positively regulates expression of the major QS regulators LasR, RhlR and MvfR, and genes of the Pseudomonas quinolone system. Chromatin immunoprecipitation (ChIP)-Seq and ChIP–quantitative real-time polymerase chain reaction studies show that AmpR binds to the ampC promoter both in the absence and presence of β-lactams. In addition, AmpR directly binds the lasR promoter, encoding the QS master regulator. Comparison of the AmpR-binding sequences from the transcriptome and ChIP-Seq analyses identified an AT-rich consensus-binding motif. This study further attests to the role of AmpR in regulating virulence and physiological processes in P. aeruginosa.

Pseudomonas aeruginosa AmpR: an acute–chronic switch regulator

Pseudomonas aeruginosa is one of the most intractable human pathogens that pose serious clinical challenge due to extensive prevalence of multidrug-resistant clinical isolates. Armed with abundant virulence and antibiotic resistance mechanisms, it is a major etiologic agent in a number of acute and chronic infections. A complex and intricate network of regulators dictates the expression of pathogenicity factors in P. aeruginosa. Some proteins within the network play key roles and control multiple pathways. This review discusses the role of one such protein, AmpR, which was initially recognized for its role in antibiotic resistance by regulating AmpC β-lactamase. Recent genomic, proteomic and phenotypic analyses demonstrate that AmpR regulates expression of hundreds of genes that are involved in diverse pathways such as β-lactam and non-β-lactam resistance, quorum sensing and associated virulence phenotypes, protein phosphorylation, and physiological processes. Finally, ampR mutations in clinical isolates are reviewed to shed light on important residues required for its function in antibiotic resistance. The prevalence and evolutionary implications of AmpR in pathogenic and nonpathogenic proteobacteria are also discussed. A comprehensive understanding of proteins at nodal positions in the P. aeruginosa regulatory network is crucial in understanding, and ultimately targeting, the pathogenic stratagems of this organism.

Role of Pseudomonas aeruginosa AmpR on β-lactam and non-β-lactam transient cross-resistance upon pre-exposure to subinhibitory concentrations of antibiotics.

Pseudomonas aeruginosa is one of the most dreaded opportunistic pathogens accounting for 10 % of hospital-acquired infections, with a 50 % mortality rate in chronically ill patients. The increased prevalence of drug-resistant isolates is a major cause of concern. Resistance in P. aeruginosa is mediated by various mechanisms, some of which are shared among different classes of antibiotics and which raise the possibility of cross-resistance. The goal of this study was to explore the effect of subinhibitory concentrations (SICs) of clinically relevant antibiotics and the role of a global antibiotic resistance and virulence regulator, AmpR, in developing cross-resistance. We investigated the induction of transient cross-resistance in P. aeruginosa PAO1 upon exposure to SICs of antibiotics. Pre-exposure to carbapenems, specifically imipenem, even at 3 ng ml(-1), adversely affected the efficacy of clinically used penicillins and cephalosporins. The high β-lactam resistance was due to elevated expression of both ampC and ampR, encoding a chromosomal β-lactamase and its regulator, respectively. Differences in the susceptibility of ampR and ampC mutants suggested non-AmpC-mediated regulation of β-lactam resistance by AmpR. The increased susceptibility of P. aeruginosa in the absence of ampR to various antibiotics upon SIC exposure suggests that AmpR plays a major role in the cross-resistance. AmpR was shown previously to be involved in resistance to quinolones by regulating MexEF-OprN efflux pump. The data here further indicate the role of AmpR in cross-resistance between quinolones and aminoglycosides. This was confirmed using quantitative PCR, where expression of the mexEF efflux pump was further induced by ciprofloxacin and tobramycin, its substrate and a non-substrate, respectively, in the absence of ampR. The data presented here highlight the intricate cross-regulation of antibiotic resistance pathways at SICs of antibiotics and the need for careful assessment of the order of antibiotic regimens as this may have dire consequences. Targeting a global regulator such as AmpR that connects diverse pathways is a feasible therapeutic approach to combat P. aeruginosa pathogenesis.

LTQ-XL mass spectrometry proteome analysis expands the Pseudomonas aeruginosa AmpR regulon to include cyclic di-GMP phosphodiesterases and phosphoproteins, and identifies novel open reading frames

UNLABELLED Pseudomonas aeruginosa is well known for its antibiotic resistance and intricate regulatory network, contributing to its success as an opportunistic pathogen. This study is an extension of our transcriptomic analyses (microarray and RNA-Seq) to understand the global changes in PAO1 upon deleting a gene encoding a transcriptional regulator AmpR, in the presence and absence of β-lactam antibiotic. This study was performed under identical conditions to explore the proteome profile of the ampR deletion mutant (PAOΔampR) using LTQ-XL mass spectrometry. The proteomic data identified ~53% of total PAO1 proteins and expanded the master regulatory role of AmpR in determining antibiotic resistance and multiple virulence phenotypes in P. aeruginosa. AmpR proteome analysis identified 853 AmpR-dependent proteins, which include 102 transcriptional regulators and 21 two-component system proteins. AmpR also regulates cyclic di-GMP phosphodiesterases (PA4367, PA4969, PA4781) possibly affecting major virulence systems. Phosphoproteome analysis also suggests a significant role for AmpR in Ser, Thr and Tyr phosphorylation. These novel mechanisms of gene regulation were previously not associated with AmpR. The proteome analysis also identified many unannotated and misannotated ORFs in the P. aeruginosa genome. Thus, our data sheds light on important virulence regulatory pathways that can potentially be exploited to deal with P. aeruginosa infections. BIOLOGICAL SIGNIFICANCE The AmpR proteome data not only confirmed the role of AmpR in virulence and resistance to multiple antibiotics, but also expanded the perimeter of AmpR regulon. The data presented here points to the role of AmpR in regulating cyclic di-GMP levels and phosphorylation of Ser, Thr and Tyr, adding another dimension to the regulatory functions of AmpR. We also identify some previously unannotated/misannotated ORFs in the P. aeruginosa genome, indicating the limitations of existing ORF analyses software. This study will contribute towards understanding complex genetic organization of P. aeruginosa. Whole genome proteomic picture of regulators at higher nodal positions in the regulatory network will not only help us link various virulence phenotypes but also design novel therapeutic strategies.

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus - a tightly cross-linked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to β-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC β-lactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.

Pseudomonas aeruginosa MifS-MifR Two-Component System Is Specific for α-Ketoglutarate Utilization.

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

Alginate-regulating genes are identified in the clinical cystic fibrosis isolate of Pseudomonas aeruginosa PA2192

Cystic fibrosis (CF) is a genetic disorder that leads to a buildup of mucus in the lungs ideal for bacterial colonization. When Pseudomonas aeruginosa enters the CF lung, it undergoes a conversion from nonmucoid to mucoid; colonization by a mucoid strain of P. aeruginosa greatly increases mortality. The mucoid phenotype is due to the production of alginate. The regulator of alginate production is the AlgT/U sigma factor. The observed phenotypic conversion is due to a mutation in the mucA gene coding for an anti-sigma factor, MucA, which sequesters AlgT/U. This mucoid phenotype is unstable when the strains are removed from the lung as they acquire second-site mutations. This in vitro reversion phenomenon is utilized to identify novel genes regulating alginate production. Previously, second-site mutations were mapped to algT/U, algO, and mucP, demonstrating their role in alginate regulation. Most of these studies were performed using a non-CF isolate. It was hypothesized that second site mutations in a clinical strain would be mapped to the same genes. In this study, a clinical, hyper-mucoid P. aeruginosa strain PA2192 was used to study the reversion phenomenon. This study found that PA2192 has a novel mucA mutation which was named them mucA180 allele. Twelve colonies were sub-cultured for two weeks without aeration at room temperature in order to obtain nonmucoid suppressors of alginate production (sap). Only 41 sap mutants were stable for more than 48 hours — a reversion frequency of 3.9% as compared to ~90% in laboratory strains showing that PA2192 has a stable mucoid phenotype. This phenotype was restored in 28 of the 41 sap mutants when complemented with plasmids harboring algT/U. Four of the sap mutants are complemented by algO. Sequence analyses of the algT/U mutants have found no mutations in the coding region or promoter leading to the hypothesis that there is another, as yet unidentified mechanism of alginate regulation in this clinical strain.

Corrigendum: Outer-membrane protein LptD (PA0595) plays a role in the regulation of alginate synthesis in Pseudomonas aeruginosa

Purpose. The presence of alginate‐overproducing (Alg+) strains of Pseudomonas aeruginosa in cystic fibrosis patients is indicative of chronic infection. The Alg+ phenotype is generally due to a mutation in the mucA gene, encoding an innermembrane protein that sequesters AlgT/U, the alginate‐specific sigma factor. AlgT/U release from the anti‐sigma factor MucA is orchestrated via a complex cascade called regulated intramembrane proteolysis. The goal of this study is to identify new players involved in the regulation of alginate production. Methodology. Previously, a mutant with a second‐site suppressor of alginate production (sap), sap27, was isolated from the constitutively Alg+ PDO300 that harbours the mucA22 allele. A cosmid from a P. aeruginosa minimum tiling path library was identified via en masse complementation of sap27. The cosmid was transposon mutagenized to map the contributing gene involved in the alginate production. The identified gene was sequenced in sap27 along with algT/U, mucA, algO and mucP. The role of the novel gene was explored using precise in‐frame algO and algW deletion mutants of PAO1 and PDO300. Results/Key findings. The gene responsible for restoring the mucoid phenotype was mapped to lptD encoding an outer‐membrane protein. However, the sequencing of sap27 revealed a mutation in algO, but not in lptD. In addition, we demonstrate that lipopolysaccharide transport protein D (LptD)‐dependent alginate production requires AlgW in PAO1 and AlgO in PDO300. Conclusion. LptD plays a specific role in alginate production. Our findings suggest that there are two pathways for the production of alginate in P. aeruginosa, one involving AlgW in the wild‐type, and one involving AlgO in the mucA22 mutant.

Otopathogenic Pseudomonas aeruginosa Enters and Survives Inside Macrophages

Otitis media (OM) is a broad term describing a group of infectious and inflammatory disorders of the middle ear. Despite antibiotic therapy, acute OM can progress to chronic suppurative otitis media (CSOM) characterized by ear drum perforation and purulent discharge. Pseudomonas aeruginosa is the most common pathogen associated with CSOM. Although, macrophages play an important role in innate immune responses but their role in the pathogenesis of P. aeruginosa-induced CSOM is not known. The objective of this study is to examine the interaction of P. aeruginosa with primary macrophages. We observed that P. aeruginosa enters and multiplies inside human and mouse primary macrophages. This bacterial entry in macrophages requires both microtubule and actin dependent processes. Transmission electron microscopy demonstrated that P. aeruginosa was present in membrane bound vesicles inside macrophages. Interestingly, deletion of oprF expression in P. aeruginosa abrogates its ability to survive inside macrophages. Our results suggest that otopathogenic P. aeruginosa entry and survival inside macrophages is OprF-dependent. The survival of bacteria inside macrophages will lead to evasion of killing and this lack of pathogen clearance by phagocytes contributes to the persistence of infection in CSOM. Understanding host–pathogen interaction will provide novel avenues to design effective treatment modalities against OM.