Induction of inflammatory cytokines and nitric oxide in J774.2 cells and murine macrophages by lipoteichoic acid and related cell wall antigens from Staphylococcus epidermidis

Staphylococcus epidermidis causes infections associated with medical devices including central venous catheters, orthopaedic prosthetic joints and artificial heart valves. This coagulase-negative Staphylococcus produces a conventional cellular lipoteichoic acid (LTA) and also releases a short-glycerophosphate-chain-length form of LTA (previously termed lipid S) into the medium during growth. The relative pro-inflammatory activities of cellular and short-chain-length exocellular LTA were investigated in comparison with peptidoglycan and wall teichoic acid from S. epidermidis and LPS from Escherichia coli O111. The ability of these components to stimulate the production of proinflammatory cytokines [interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α] and nitric oxide was investigated in a murine macrophage-like cell line (J774.2), and in peritoneal and splenic macrophages. On a weight-for-weight basis the short-chain-length exocellular LTA was the most active of the S. epidermidis products, stimulating significant amounts of each of the inflammatory cytokines and nitric oxide, although it was approximately 100-fold less active than LPS from E. coli. By comparison the full-chain-length cellular LTA and peptidoglycan were less active and the wall teichoic acid had no activity. As an exocellular product potentially released from S. epidermidis biofilms, the short-chain-length exocellular LTA may act as the prime mediator of the host inflammatory response to device-related infection by this organism and act as the Gram-positive equivalent of LPS in Gram-negative sepsis. The understanding of the role of short-chain-length exocellular LTA in Gram-positive sepsis may lead to improved treatment strategies. © 2005 SGM.

Automated synthesis and evaluation of potential new anti-microbial agents

A series of N1-benzylideneheteroarylcarboxamidrazones was prepared in an automated fashion, and tested against Mycobacterium fortuitum in a rapid screen for antimycobacterial activity. Many of the compounds from this series were also tested against Mycobacterium tuberculosis, and the usefulness as M.fortuitum as a rapid, initial screen for anti-tubercular activity evaluated. Various deletions were made to the N1-benzylideneheteroarylcarboxamidrazone structure in order to establish the minimum structural requirements for activity. The N1-benzylideneheteroarylcarbox-amidrazones were then subjected to molecular modelling studies and their activities against M.fortuitum and M.tuberculosis were analysed using quantitative structure-analysis relationship (QSAR) techniques in the computational package TSAR (Oxford Molecular Ltd.). A set of equations predictive of antimycobacterial activity was hereby obtained. The series of N1-benzylidenehetero-arylcarboxamidrazones was also tested against a multidrug-resistant strain of Staphylococcus aureus (MRSA), followed by a panel of Gram-positive and Gram-negative bacteria, if activity was observed for MRSA. A set of antimycobacterial N1-benzylideneheteroarylcarboxamidrazones was hereby discovered, the best of which had MICs against m. fortuitum in the range 4-8μgml-1 and displayed 94% inhibition of M.tuberculosis at a concentration of 6.25μgml-1. The antimycobacterial activity of these compounds appeared to be specific, since the same compounds were shown to be inactive against other classes of organisms. Compounds which were found to be sufficiently active in any screen were also tested for their toxicity against human mononuclear leucocytes. Polyethylene glycol (PEG) was used as a soluble polymeric support for the synthesis of some fatty acid derivatives, containing an isoxazoline group, which may inhibit mycolic acid synthesis in mycobacteria. Both the PEG-bound products and the cleaved, isolated products themselves were tested against M.fortuitum and some low levels of antimycobacterial activity were observed, which may serve as lead compounds for further studies.

Sensitivity of staphylococcus epidermidis to chlorhexidine and associated resistance properties

Staphylococcus epidermidis are common Gram-positive bacteria and are responsible for a number of life-threatening nosocomial infections. Treatment of S. epidermidis infection is problematic because the organism is usually resistant to many antibiotics. The high degree of resistance of this organism to a range of antibiotics and disinfectants is widely known. The aims of this thesis were to investigate and evaluate the susceptibility of isolates of S. epidermidis from various infections to chlorhexidine (CHX) and to other disinfectants such as benzalkonium chloride (BKC), triclosan (TLN) and povidone-iodine (PI). In addition, the mechanisms of resistance of S. epidermidis to chlorhexidine (the original isolates and strains adapted to chlorhexidine by serial passage) were examined and co-resistance to clinically relevant antibiotics investigated. In 3 of the 11 S. epidermidis strains passaged in increasing concentrations of chlorhexidine, resistance to the disinfectant arose (16-fold). These strains were examined further, each showing stable chlorhexidine resistance. Co-resistance to other disinfectants such as BKC, TLN and PI and changes in cell surface hydrophobicity were observed. Increases in resistance were accompanied by an increase in the proportion of neutral lipids and phospholipids in the cell membrane. This increase was most marked in diphosphatidylglycerol. These observations suggest that some strains of S. epidermidis can become resistant to chlorhexidine and related disinfectants/antiseptics by continual exposure. The mechanisms of resistance appear to be related to changes in membrane lipid compositions.

Serodiagnosis of staphylococcal sepsis

The coagulase-negative staphylococci are the most frequent cause of sepsis associated with indwelling intravascular catheters. Current microbiological investigations to support the diagnosis of catheter-related sepsis (CRS) include the culture of blood and catheter tips, however positive results may reflect specimen contamination, or colonisation of the catheter rather than true sepsis. Previous serological approaches to assist in the diagnosis of CRS based on cellular staphylococcal antigens have been of limited value. In this current study, the serodiagnostic potential of an exocellular antigen produced by 7 strains of coagulase-negative staphylococci cultured in brain heart infusion broth was investigated. Antigenic material isolated by gel permeation from liquid culture was characterised by immunological techniques and chemical analysis. Characterisation of the exocellular antigen revealed a novel glycerophosphoglycolipid, termed lipid S. which shared antigenic determinants with lipoteichoic acid, but differed by comprising a glycerophosphate chain length of only 6 units. In addition, lipid S was immunologically distinct from diphosphatidyl glycerol, a constituent cell membrane phospho lipid. An indirect enzyme linked immunosorbent assay (ELISA) based on lipid S was subsequently developed and used to determine serum antibody levels (IgM and IgG) in 67 patients with CRS due to staphylococci, and 67 patients with a central venous catheter (CVC) in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the lipid S IgG ELISA was 75% and 90% respectively whilst the IgM assay had sensitivity and specificity of 52% and 85%. The addition of GullSORereagent to the EL1SA procedure to remove competing serum IgG and rheumatoid factor did not significantly improve the performance of the IgM assay. The serological response in serial serum samples of 13 patients with CRS due to staphylococci was investigated. Elevated levels of antibody were detected at an early stage of infection, prior to the isolation of microorganisms by standard culture methods, and before the clinical presentation of sepsis in 3 patients. The lipid S ELISA was later optimised and a rapid 4-hour assay developed for the serodiagnosis of CRS. Serum IgG levels were determined in 40 patients with CRS due to staphylococci and 40 patients with a CVC in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the rapid IgG assay was 70% and 100% respectively. Elevated serum antibody levels in patients with endocarditis, prosthetic joint infection and pyogenic spondylodiscitis due to Gram-positive cocci were also detected with the lipid S ELISA suggesting that the assay may facilitate the diagnosis of these infections. Unexpected increased levels of anti-lipid S IgG in 31% of control patients with sciatica suggested a possible microbial aetiology of this condition. Further investigation of some of these patients by culture of microdiscectomy tissue removed at operation, revealed the presence of low-virulent microorganisms in 37% of patients of which Propionibacterium aeries accounted for 85% of the positive culture isolates. The results suggested a previously unrecognised association between P. acnes and sciatica, which may have implications for the future management of the condition.

Peptidoglycan derived from Staphylococcus epidermidis induces Connexin43 hemichannel activity with consequences on the innate immune response in endothelial cells

Gram-positive bacterial cell wall components including PGN (peptidoglycan) elicit a potent pro-inflammatory response in diverse cell types, including endothelial cells, by activating TLR2 (Toll-like receptor 2) signalling. The functional integrity of the endothelium is under the influence of a network of gap junction intercellular communication channels composed of Cxs (connexins) that also form hemichannels, signalling conduits that are implicated in ATP release and purinergic signalling. PGN modulates Cx expression in a variety of cell types, yet effects in endothelial cells remain unresolved. Using the endothelial cell line b.End5, a 6 h challenge with PGN induced IL-6 (interleukin 6), TLR2 and Cx43 mRNA expression that was associated with enhanced Cx43 protein expression and gap junction coupling. Cx43 hemichannel activity, measured by ATP release from the cells, was induced following 15 min of exposure to PGN. Inhibition of hemichannel activity with carbenoxolone or apyrase prevented induction of IL-6 and TLR2 mRNA expression by PGN, but had no effect on Cx43 mRNA expression levels. In contrast, knockdown of TLR2 expression had no effect on PGN-induced hemichannel activity, but reduced the level of TLR2 and Cx43 mRNA expression following 6 h of PGN challenge. PGN also acutely induced hemichannel activity in HeLa cells transfected to express Cx43, but had no effect in Cx43-deficient HeLa OHIO cells. All ATP responses were blocked with Cx-specific channel blockers. We conclude that acute Cx43 hemichannel signalling plays a role in the initiation of early innate immune responses in the endothelium.

Association between sciatica and Propionibacterium acnes

We hypothesised that the inflammation seen around the nerve root in patients with sciatica may be caused by microbial infection. We used a newly developed serological test to diagnose deep-seated infections caused by low virulent gram-positive microorganisms. 43 of 140 (31%) patients with sciatica tested positive. Intervertebral disc material from a further 36 patients with severe sciatica who had undergone microdiscectomy was cultured for the presence of microorganisms. 19 of these patients (53%) had positive cultures after long-term incubation. Propionibacterium acnes was isolated from 16 of the 19 (84%) positive samples. Low virulent microorganisms, in particular P acnes, might be causing a chronic low-grade infection in the lower intervertebral discs of patients with severe sciatica. These microorganisms could have gained access to the spinal disc after previous minor trauma.

Surface microbial consortia from Livarot, a French smear-ripened cheese

The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR (rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG) 55-PCR, and when appropriate by 16S rDNA sequencing and SDS-PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly assigned to the genera Arthrobacter, Brevibacterium, Corynebacterium, and Staphylococcus. New taxa related to the genera Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed a high level of diversity and mainly included members of the genera Alcaligenes, Hafnia, Proteus, Pseudomonas, and Psychrobacter. Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gramnegative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of their positive versus negative role should be objectively examined.

Propionibacterium acnes:infection beyond the skin

Propionibacterium acnes is a Gram-positive bacterium that forms part of the normal flora of the skin, oral cavity, large intestine, the conjunctiva and the external ear canal. Although primarily recognized for its role in acne, P. acnes is an opportunistic pathogen, causing a range of postoperative and device-related infections. These include infections of the bones and joints, mouth, eye and brain. Device-related infections include those of joint prostheses, shunts and prosthetic heart valves. P. acnes may play a role in other conditions, including inflammation of the prostate leading to cancer, SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome, sarcoidosis and sciatica. If an active role in these conditions is established there are major implications for diagnosis, treatment and protection. Genome sequencing of the organism has provided an insight into the pathogenic potential and virulence of P. acnes. © 2011 Expert Reviews Ltd.

Biological activities of compounds from Piper species

It is well established that secondary metabolites play an important role in plant chemical defense. In an effort to find natural herbicides research on plant growth regulatory activity of secondary metabolites has received more and more attention recently. The genus Piper has been an important source for useful secondary metabolites.^ Crude extracts from Piper species inhibited gram-positive bacteria and higher plant growth under laboratory conditions. Bioassay-guided isolation and purification lead to the identification of safrole, a phenylpropene, as the responsible agent for the inhibitory activity. Safrole was found to leach from naturally fallen leaves with water. Mechanisms of plant growth inhibition by safrole were investigated. Disassociation of cell membrane from cell walls was determined to be a major cause.^ Phenylpropenes structurally similar to safrole had similar phytogrowth inhibitory activity. It is postulated that phenylpropanoids are an important group of naturally occurring secondary metabolites in plant-plant interactions. ^

The 4-aza-S-ribosyl-L-homocysteine derivatives and the related gamma-lactam and azahemiacetal analogs: Synthesis, inhibition and quorum sensing activity

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence, critical for establishing infection. There are two major pathways of QS systems. Type 1 is species specific or intra-species communication in which N-acylhomoserine lactones (Gram-negative bacteria) or oligopeptides (Gram-positive bacteria) are employed as signaling molecules (autoinducer one). Type 2 is inter-species communication in which S-4,5-dihydroxy-2,3-pentanedione (DPD) or its borate esters are used as signaling molecules. The DPD is biosynthesized by LuxS enzyme from S-ribosylhomocysteine (SRH). Recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of new generation of antibacterial agents. Interruption of QS by small molecules is one of the viable options as it does not affect bacterial growth but only virulence, leading to less incidence of microbial resistance. Thus, in this work, inhibitors of both N-acylhomoserine lactone (AHL) mediated intra-species and LuxS enzyme, involved in inter-species QS are targeted. The γ-lactam and their reduced cyclic azahemiacetal analogs, bearing the additional alkylthiomethyl substituent, were designed and synthesized targeting AHL mediated QS systems in P. aeruginosa and Vibrio harveyi. The γ-lactams with nonylthio or dodecylthio chains acted as inhibitors of las signaling in P. aeruginosa with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent were found to strongly inhibit both las and rhl signaling in P. aeruginosa at higher concentrations. However, lactam and their azahemiacetal analogs were found to be inactive in V. harveyi QS systems. The 4-aza-S-ribosyl-L-homocysteine (4-aza-SRH) analogs and 2-deoxy-2-substituted-S-ribosyl-L-homocysteine analogs were designed and synthesized targeting Bacillus subtilis LuxS enzyme. The 4-aza-SRH analogs in which oxygen in ribose ring is replaced by nitrogen were further modified at anomeric position to produce pyrrolidine, lactam, nitrone, imine and hemiaminal analogs. Pyrrolidine and lactam analogs which lack anomeric hydroxyl, acted as competitive inhibitors of LuxS enzyme with KI value of 49 and 37 µM respectively. The 2,3-dideoxy lactam analogs were devoid of activity. Such findings attested the significance of hydroxyl groups for LuxS binding and activity. Hemiaminal analog of SRH was found to be a time-dependent inhibitor with IC50 value of 60 µM.

Diversity of Vancomycin Genes in Sub-tropical Soils

With the increased antibiotic exposure from anthropogenic sources, soil microbes are an ever-increasing ecological pool of resistant bacteria. This is the case with bacterial resistance to vancomycin through transfer of van-resistance genes by transposons. Studies show that bacterial species other than enteroccoci harbor genetic-like elements such as the Tn1546 transposon containing vancomycin-resistant genes. Overuse and misuse of antibiotics in hospital settings and agricultural practices have led to an increase in transferability of vancomycin-resistant genes among microbes. The objective of this project is to analyze the diversity of these genes found in the soil microbes from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine the degree of resistance. Results showed that all bacterial isolates were resistant to penicillin at the 10 µg concentration and most were susceptible to varying vancomycin concentrations (10 µg, 20 µg, and 30 µg). A 1465 bp fragment was amplified from the 16S rDNA gene using 27F and 1492R universal primers from the multi-antibiotic resistant bacteria and sequenced to identify the isolates. Three Gram-negative bacteria genera were identified with the closest phylogenetic match to: Pseudomonas sp., Stenotrophomonas sp., Xanthomonas sp., as well as two Gram-positive bacteria genera: Bacillus sp. and Brevibacillus sp. The isolates’ vanA and vanB genes were amplified using the respective primers. Ongoing work is underway to sequence and compare these known van resistant genes, with the goal of revealing intrinsic vancomycin resistance present in soil bacteria.

Impacto na epidemiologia da sepse tardia após a mudança do protocolo de antimicrobianos em uma UTIN de um serviço universitário

Advances in neonatology resulted in reducing the mortality rate and the consequent increase in survival of newborn pre terms (PTN). On the other hand, there was also a considerable increase in the risk of developing health care-related infection (HAI) in its most invasive, especially for bloodstream. This situation is worrying, and prevent the occurrence of it is a challenge and becomes one of the priorities in the Neonatal Intensive Care Unit (NICU). Sepsis is the main cause of death in critical neonates and affects more than one million newborns each year, representing 40% of all deaths in neonates. The incidence of late sepsis can reach 50% in NICUs. Currently the major responsible for the occurrence of sepsis in developed countries is the coagulase negative Staphylococcus (CoNS), followed by S. aureus. The cases of HAIs caused by resistant isolates for major classes of antimicrobial agents have been increasingly frequent in the NICU. Therefore, vancomycin has to be prescribed more frequently, and, today, the first option in the treatment of bloodstream infections by resistant Staphylococcus. The objectives of this study were to assess the impact on late sepsis in epidemiology III NICU after the change of the use of antimicrobials protocol; check the frequency of multiresistant microorganisms; assess the number of neonates who came to death. This study was conducted in NICU Level III HC-UFU. three study groups were formed based on the use of the proposed late sepsis treatment protocol, with 216 belonging to the period A, 207 B and 209 to the C. The work was divided into three stages: Period A: data collected from neonates admitted to the unit between September 2010 to August 2011. was using treatment of late sepsis: with oxacillin and gentamicin, oxacillin and amikacin, oxacillin and cefotaxime. Period B: data were collected from March 2012 to February 2013. Data collection was started six months after protocol change. Due to the higher prevalence of CoNS, the initial protocol was changed to vancomycin and cefotaxime. Period C: data were collected from newborns inteerne in the unit from September 2013 to August 2014. Data collection was started six months after the protocol change, which occurred in March 2013. From the 632 neonates included in this study, 511 (80,8%) came from the gynecology and obstetrics department of the HC-UFU. The mean gestational age was 33 weeks and the prevailing sex was male (55,7%). Seventy-nine percent of the studied neonates were hospitalized at the NICU HC-UFU III because of complications related to the respiratory system. Suspicion of sepsis took to hospitalization in the unit of 1,9% of newborns. In general, the infection rate was 34,5%, and the most frequent infectious sepsis syndrome 81,2%. There was a tendency to reduce the number of neonates who died between periods A 11 and C (p = 0,053). From the 176 cases of late sepsis, 73 were clinical sepsis and 103 had laboratory confirmation, with greater representation of Gram positive bacteria, which corresponded to 67.2% of the isolates and CoNS the most frequent micro-organism (91,5%). There was a statistically significant difference in the reduction of isolation of Gram positive microorganisms between periods A and C (p = 0,0365) as well as in reducing multidrug-resistant CoNS (A and B period p = 0,0462 and A and C period, p = 0,158). This study concluded that: the CoNS was the main microorganism responsible for the occurrence of late sepsis in neonates in the NICU of HC-UFU; the main risk factors for the occurrence of late sepsis were: birth weight <1500 g, use of PICC and CUV, need for mechanical ventilation and parenteral nutrition, SNAPPE> 24 and length of stay more than seven days; the new empirical treatment protocol late sepsis, based on the use of vancomycin associated cefepime, it was effective, since promoted a reduction in insulation CoNS blood cultures between the pre and post implementation of the Protocol (A and C, respectively); just as there was a reduction in the number of newborns who evolved to death between periods A and C.

Prospecção química e avaliação biológica de Banisteriopsis laevifolia (Malpighiaceae)

The Banisteriopsis genus is widespread in traditional medicine. This work aims to contribute with information about the chemical composition and on the evaluation of the biological activity of the essential oil, the ethanol extract of the leaves and partitions of the Banisteriopsis laevifolia. The phytochemical screeningtest of ethanol extract and partitions of leaves indicated the presence of flavonoids, terpenoids, saponins, phenols and steroids compounds. Nitrogenous compounds, characteristic of some species of this family, were not detected. Flavonoids were the predominant metabolite, with the highest concentrations on the partitions ethyl acetate and n-butanol. The antibacterial activity, antifungal and cytotoxicity of the essetial oil, ethanol extract and partitions were assyed by microdilution broth method (MBM), where the minimum inhibitory concentrations (MIC) were calculated. The ethanol extract and partitions did not inhibit growth against to Gram positive bacteria tested, with MIC less than 400 mg L-1. For the Gram negative bacteria tested, the hexane and hydroethanol partitios were more effective against F. nucleatum bacteria (MIC 100 ug mL-1). The ethanol extract showed antifungal activity with MIC of 31.2 mg L-1. Ethyl acetate and n-butanol partitions showed MIC 187.5 mg L-1 and 93.7 mg L-1, respectively, arousing interest for isolation studies. The antioxidant activity was evaluated by the DPPH free radical method. The ethanolic extract, ethyl acetate and n-butanol partitions were active, since they showed EC50 values (4.53 ug mL-1, 4.07 and 8.39 ug mL-1, respectively), values equivalent to the BHT (7.3 mg L-1). The analysis by HPLC-MS/MS of the most active fractions (ethyl acetate and n-butanol) identified phenolic compounds (flavonols and phenolic acids) which exert recognized biological activity. The GC-MS analysis of the essential oils from leaves collected in two periods studied (dry and wet), showed a small variation in the number of compounds. The major classes identified for the oil collected in the dry period were aliphatic alcohols (23,4%), terpenoids (18.7%), sterols (10.4%) and long-chain alkanes (9.2%) compounds. Terpenoids (26.8%) were the major class for the rain season. The major compounds (3Z) -hexenol, phytol and untriacontano are present in the two seasons but in different amounts (19.4%, 9.8% and 7.5% during the dry season, and 17.0 %, 14.9% and 15.3% in the rainy season, respectively). The essential oil from rainy season was not effective against to the oral bacteria Gram positive and Gram negative tested. However, showed significant antifungal activity with MIC 1000 mg L-1 against Candidas. Thus, the promising results with respect to biological assays of ethanolic extract and partitions from B. laevifolia contributed to the chemical and biological knowledge of the species B. laevifolia.

Chitosan gel film bandages: correlating structure, composition, and antimicrobial properties

Chitosan gel films were successfully obtained by evaporation cast from chitosan solutions in aqueous acidic solutions of organic acids (lactic and acetic acid) as gel film bandages, with a range of additives that directly influence film morphology and porosity. We show that the structure and composition of a wide range of 128 thin gel films, is correlated to the antimicrobial properties, their biocompatibility and resistance to biodegradation. Infrared spectroscopy and solid-state 13C nuclear magnetic resonance spectroscopy was used to correlate film molecular structure and composition to good antimicrobial properties against 10 of the most prevalent Gram positive and Gram negative bacteria. Chitosan gel films reduce the number of colonies after 24 h of incubation by factors of ∼105–107 CFU/mL, compared with controls. For each of these films, the structure and preparation condition has a direct relationship to antimicrobial activity and effectiveness. These gel film bandages also show excellent stability against biodegradation with lysozyme under physiological conditions (5% weight loss over a period of 1 month, 2% in the first week), allowing use during the entire healing process. These chitosan thin films and subsequent derivatives hold potential as low-cost, dissolvable bandages, or second skin, with antimicrobial properties that prohibit the most relevant intrahospital bacteria that infest burn injuries.

Biochemical and Structural Studies on PrfA, the Transcriptional Regulator of Virulence in Listeria monocytogenes

Abstract

Listeria monocytogenes is a gram-positive soil saprophytic bacterium that is capable of causing fatal infection in humans. The main virulence regulator PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the expression of essential proteins required for host cell invasion and cell-to-cell spread. The mechanism of PrfA activation and the identity of its small molecule coactivator have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene expression by binding cAMP, increasing the DNA binding affinity of the protein and causing a significant DNA bend that facilitates RNA polymerase binding and downstream gene activation. Our data suggests PrfA activates virulence protein expression through a mechanism distinct from the canonical Crp activation mechanism that involves a combination of cysteine residue reduction and glutathione (GSH) binding.

Listeria lacking glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly, Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine (Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double mutant strains are avirulent. DNA-binding affinity, measured through fluorescence polarization assays, indicate reduction of the cysteine side chains is sufficient to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar affinity. Oxidized PrfA binds the promoters poorly.

Unexpectedly, Quad also binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH at physiologically relevant and comparable affinities, however GSH did not affect DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type PrfA differ structurally upon binding GSH, which supports the in vivo difference in infection between the regulator and its mutant.

Structures of PrfA in complex with cognate DNA, determined through X-ray crystallography, further support the disparity between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.

From these results, we hypothesize a two-step activation mechanism wherein PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular release and binds DNA, causing a slight bend in the promoter and small increase in transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24 likely visualize this intermediate complex. Increasing concentrations of GSH shift the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and is hypothesized to resemble closely the transcriptionally active structure of the cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient in this second step, which explains the decrease in virulence and implicates the cysteine residues as critical for transcription efficiency. Further structural and biochemical studies are on-going to clarify this mechanism of activation.