Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections. © 2009 Piuri et al

A Novel Inactivated Intranasal Respiratory Syncytial Virus Vaccine Promotes Viral Clearance without Th2 Associated Vaccine-Enhanced Disease

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960's led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations.In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines.These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology

Positive feedback and noise activate the stringent response regulator Rel in mycobacteria

Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator, rel. The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a "window of opportunity" in time preceding the stationary phase. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of "heterogeneity with an advantage" in mycobacteria.Comment: Accepted for publication in PLoS On

Enterohemorrhagic E. coli Requires N-WASP for Efficient Type III Translocation but Not for EspFU-Mediated Actin Pedestal Formation

Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspFU (aka TccP) that trigger the formation of F-actin-rich ‘pedestals’ beneath bound bacteria. EspFU is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspFU into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspFU, we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspFU were the only bacterial effectors required for pedestal formation, and the EspFU sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspFU, presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization

Conditional Gene Expression in Mycobacterium abscessus

Mycobacterium abscessus is an emerging human pathogen responsible for lung infections, skin and soft-tissue infections and disseminated infections in immunocompromised patients. It may exist either as a smooth (S) or rough (R) morphotype, the latter being associated with increased pathogenicity in various models. Genetic tools for homologous recombination and conditional gene expression are desperately needed to allow the study of M. abscessus virulence. However, descriptions of knock-out (KO) mutants in M. abscessus are rare, with only one KO mutant from an S strain described so far. Moreover, of the three major tools developed for homologous recombination in mycobacteria, only the one based on expression of phage recombinases is working. Several conditional gene expression tools have recently been engineered for Mycobacterium tuberculosis and Mycobacterium smegmatis, but none have been tested yet in M. abscessus. Based on previous experience with genetic tools allowing homologous recombination and their failure in M. abscessus, we evaluated the potential interest of a conditional gene expression approach using a system derived from the two repressors system, TetR/PipOFF. After several steps necessary to adapt TetR/PipOFF for M. abscessus, we have shown the efficiency of this system for conditional expression of an essential mycobacterial gene, fadD32. Inhibition of fadD32 was demonstrated for both the S and R isotypes, with marginally better efficiency for the R isotype. Conditional gene expression using the dedicated TetR/PipOFF system vectors developed here is effective in S and R M. abscessus, and may constitute an interesting approach for future genetic studies in this pathogen

An Oligopeptide Transporter of Mycobacterium tuberculosis Regulates Cytokine Release and Apoptosis of Infected Macrophages

Background: The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppA(MTB). Methodology/Principal Findings: OppA(MTB) was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppA(MTB) confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1 beta, IL-6 and TNF-alpha was also compromised after inactivation of oppD. Conclusions: Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis

Induction of IgG3 to LPS via Toll-Like Receptor 4 Co-Stimulation

B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR) and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs) also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise

Profiling of Differentially Expressed Genes Using Suppression Subtractive Hybridization in an Equine Model of Chronic Asthma

Background :\ud Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma.\ud \ud Objective :\ud To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition.\ud \ud Methods :\ud Eleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay.\ud \ud Results :\ud Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways.\ud \ud Conclusions :\ud Pathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies

Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans