Sandeep Tamber

Role of the Novel OprD Family of Porins in Nutrient Uptake in Pseudomonas aeruginosa

To circumvent the permeability barrier of its outer membrane, Pseudomonas aeruginosa has evolved a series of specific porins. These channels have binding sites for related classes of molecules that facilitate uptake under nutrient-limited conditions. Here, we report on the identification of a 19-member family of porins similar to the basic-amino-acid-specific porin OprD. The members of this family fell into one of two phylogenetically distinct clusters, one bearing high similarity to OprD and the other bearing most similarity to the putative phenylacetic acid uptake porin PhaK of Pseudomonas putida. Analysis of the genome context, operon arrangement, and regulation of the PhaK-like porin OpdK indicated that it might be involved in vanillate uptake. This result was confirmed by demonstrating that an opdK mutant had a deficiency in the ability to grow on vanillate as a carbon source. To extrapolate these data to other paralogues within this family, the substrate specificities of 6 of the 17 remaining OprD homologues were inferred using an approach similar to that used with opdK. The specificities determined were as follows: OpdP, glycine-glutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH, cis-aconitate; and OpdO, pyroglutamate. Thus, members of the OprD subfamily took up amino acids and related molecules, and those characterized members most similar to PhaK were responsible for the uptake of a diverse array of organic acids. These results imply that there is a functional basis for the phylogenetic clustering of these proteins and provide a framework for studying OprD homologues in other organisms.

MgrA Represses Biofilm Formation in Staphylococcus aureus

ABSTRACT MgrA is a pleiotropic regulator that controls autolysis, virulence, and efflux pump activity in Staphylococcus aureus. We recently found that mgrA mutants of strains RN6390, SH1000, and MW2 also displayed enhanced biofilm formation compared with their respective parents. The biofilms formed by mgrA mutants of RN6390 and MW2 are independent of sigB and ica loci, two genetic elements that have been previously associated with biofilm formation in S. aureus. Biofilms formed by mgrA mutants are dependent on the expression of surface proteins mediated by the sortase gene srtA. Extracellular DNA was also a crucial component of the early biofilm of mgrA mutants. Genetic analysis indicated that biofilm formation in mgrA mutants is mediated in part by agr RNAIII, a genetic locus regulated by mgrA. Additionally, SarA is important to biofilm formation in mgrA mutants since the double sarA mgrA mutants failed to form biofilms compared to single mgrA mutants of RN6390 and MW2. However, the SarA-mediated effect is independent of agr and proteases such as V8 protease and aureolysin. Collectively, our data showed MgrA to be a repressor of biofilm formation, and biofilms formed by mgrA mutants have features that are distinct from other reported biofilm types in S. aureus.

Role of PknB Kinase in Antibiotic Resistance and Virulence in Community-Acquired Methicillin-Resistant Staphylococcus aureus Strain USA300

ABSTRACT The regulation of cellular processes by eukaryote-like serine/threonine kinases is widespread in bacteria. In the last 2 years, several studies have examined the role of serine/threonine kinases in Staphylococcus aureus on cell wall metabolism, autolysis, and virulence, mostly in S. aureus laboratory isolates in the 8325-4 lineage. In this study, we showed that the pknB gene (also called stk1) of methicillin-resistant S. aureus (MRSA) strain COL and the community-acquired MRSA (CA-MRSA) strain USA300 is involved in cell wall metabolism, with the pknB mutant exhibiting enhanced sensitivity to β-lactam antibiotics but not to other classes of antibiotics, including aminoglycosides, ciprofloxacin, bactrim, and other types of cell wall-active agents (e.g., vancomycin and bacitracin). Additionally, the pknB mutant of USA300 was found to be more resistant to Triton X-100-induced autolysis and also to lysis by lysostaphin. We also showed that pknB is a positive regulator of sigB activity, resulting in compromise in its response to heat and oxidative stresses. In association with reduced sigB activity, the expression levels of RNAII and RNAIII of agr and the downstream effector hla are upregulated while spa expression is downmodulated in the pknB mutant compared to the level in the parent. Consistent with an enhanced agr response in vitro, virulence studies of the pknB mutant of USA300 in a murine cutaneous model of infection showed that the mutant was more virulent than the parental strain. Collectively, our results have linked the pknB gene in CA-MRSA to antibiotic resistance, sigB activity, and virulence and have highlighted important differences in pknB phenotypes (virulence and sigB activity) between laboratory isolates and the prototypic CA-MRSA strain USA300.

The major outer membrane protein OprG of Pseudomonas aeruginosa contributes to cytotoxicity and forms an anaerobically regulated, cation-selective channel

OprG of Pseudomonas aeruginosa is a member of the very large and widely distributed but poorly characterized OmpW (PF0392) family of outer membrane proteins. It was established here that OprG was highly transcribed in anaerobic environments rich in iron via the ANR regulator. In the absence of OprG, P. aeruginosa was significantly less cytotoxic toward human bronchial epithelial cells. Planar bilayer studies indicated that purified OprG formed cationic-selective channels with a conductance of 500 pS in 1 M KCl; however, contrary to previous reports, OprG did not appear to be involved in either iron or antibiotic uptake.

SarZ Promotes the Expression of Virulence Factors and Represses Biofilm Formation by Modulating SarA and agr in Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a remarkably adaptable organism capable of multiple modes of growth in the human host, as a part of the normal flora, as a pathogen, or as a biofilm. Many of the regulatory pathways governing these modes of growth are centered on the activities of two regulatory molecules, the DNA binding protein SarA and the regulatory RNAIII effector molecule of the agr system. Here, we describe the modulation of these regulators and their downstream target genes by SarZ, a member of the SarA/MarR family of transcriptional regulators. Transcriptional and phenotypic analyses of a sarZ mutant demonstrated that the decreased transcription of mgrA and the agr RNAIII molecule was accompanied by increased transcription of spa (protein A) and downregulation of hla (alpha-hemolysin) and sspA (V8 protease) transcripts when compared to its isogenic parent. The decrease in protease activity was also associated with an increase in SarA expression. Consistent with an increase in SarA levels, the sarZ mutant displayed an enhanced ability to form biofilms. Together, our results indicate that SarZ may be an important regulator governing the dissemination phase of S. aureus infections, as it promotes toxin expression while repressing factors required for biofilm formation.

Characterization of OpdH, a Pseudomonas aeruginosa Porin Involved in the Uptake of Tricarboxylates

The Pseudomonas aeruginosa outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of cis-aconitate. Here we demonstrate that this porin may also enable P. aeruginosa to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the opdH gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions.

The Staphylococcus-Specific Gene rsr Represses agr and Virulence in Staphylococcus aureus

ABSTRACT The expression of virulence factors in Staphylococcus aureus is tightly coordinated by a vast network of regulatory molecules. In this report, we characterize a genetic locus unique to staphylococci called rsr that has a role in repressing two key virulence regulators, sarR and agr. Using strain SH1000, we showed that the transcription of virulence effectors, such as hla, sspA, and spa, is altered in an rsr mutant in a way consistent with agr upregulation. Analysis of RNAIII expression of the agr locus in rsr and rsr-sarR mutants indicated that rsr likely contributes to agr expression independently of SarR. We also provide evidence using a murine model of S. aureus skin infection that the effects mediated by rsr reduce disease progression.

The Outer Membranes of Pseudomonads

The study of bacterial cell surfaces began in 1675 when Leeuwenhoek peering through his microscope wondered what “held [bacteria] together, or what contained them”92. Since that time, our knowledge of cell surfaces, namely the Gram-negative outer membrane, has grown considerably. In addition to containing the bacteria, the outer membrane mediates a myriad of functions including other structural roles such as maintaining bacterial shape and providing a scaffold for fimbriae and flagella. Another key function of the outer membrane is to mediate the interactions between Gram-negative bacteria and their environment, primarily by determining which compounds enter and exit the cell. This complex task involves the integration of lipidic components involved in the barrier function of the outer membrane and proteins involved in the uptake and efllux of the various compounds able to traverse this barrier.

Antibiotic Resistance Due to Reduced Uptake

The introduction of antibiotic therapy for the treatment of bacterial infections has led to a greatly increased human life span compared to that in the pre-antibiotic era. However, a disturbing trend has also been noted in that, within a very short period of time following the introduction of a new antibiotic, resistance to that antibiotic begins to emerge, a factor that is becoming increasingly meaningful as the discovery of new antibiotics wanes [1–3]. There are a number of mechanisms by which a bacterium may become resistant to a particular antibiotic. Generally these include, but are not limited to, modification of the drug to render it inactive, modification of the drug target, such that it is incapable of interacting with the drug and decreased uptake of the antibiotic into the cell, due to reduced transport and/or increased efflux. Recent functional genomic studies have also implied that antibiotics may have more complex mechanisms of action than first thought and we are beginning to appreciate that in addition to the mutation of primary targets, subtle mutations in secondary targets are likely to be influential [4, 5]. Moreover, a growing body of evidence suggests that the temporary changes in susceptibility associated with the phenomenon of adaptive resistance may also be important for the global rise in bacterial resistance to antimicrobial compounds [6]. This chapter will focus on the contribution of a decreased antibiotic uptake to an increase in antibacterial resistance.