Metabolic analyses reveal common adaptations in two invasive Haemophilus influenzae strains

Non-typeable Haemophilus influenzae (NTHi) is a major pathogen in upper and lower respiratory tract infections in humans, and is increasingly also associated with invasive disease. We have examined two unrelated NTHi invasive disease isolates, R2866 and C188, in order to identify metabolic and physiological properties that distinguish them from respiratory tract disease isolates such as Hi2019. While the general use of the Hi metabolic network was similar across all three strains, the two invasive isolates secreted increased amounts of succinate which can have anti-inflammatory properties. In addition, they showed a common shift in their carbon source utilization patterns, with strongly enhanced metabolism of nucleoside substrates, glucose and sialic acid. The latter two are major compounds present in blood and CSF. Interestingly, C188 and R2866 also shared a reduced ability to invade or survive intracellularly in 16HBE14 bronchial epithelial cells relative to Hi2019 (4-fold (4 h), 25-fold (24 h) reduction). Altered metabolic properties such as the ones observed here could arise from genomic adaptations that NTHi undergo during infection. Together these data indicate that shifts in substrate preferences in otherwise conserved metabolic pathways may underlie strain niche specificity and thus have the potential to alter the outcomes of host-NTHi interactions

Control of Bacterial Sulfite Detoxification by Conserved and Species-Specific Regulatory Circuits

Although sulfite, a by-product of the degradation of many sulfur compounds, is highly reactive and can cause damage to DNA, proteins and lipids, comparatively little is known about the regulation of sulfite-oxidizing enzyme (SOEs) expression. Here we have investigated the regulation of SOE-encoding genes in two species of α-Proteobacteria, Sinorhizobium meliloti and Starkeya novella, that degrade organo- and inorganic sulfur compounds, respectively, and contain unrelated types of SOEs that show different expression patterns. Our work revealed that in both cases, the molecular signal that triggers SOE gene expression is sulfite, and strong up-regulation depends on the presence of a sulfite-responsive, cognate Extracytoplasmic function (ECF) sigma factor, making sulfite oxidation a bacterial stress response. An additional RpoE1-like ECF sigma factor was also involved in the regulation, but was activated by different molecular signals, taurine (Sm) and tetrathionate (Sn), respectively, targeted different gene promoters, and also differed in the magnitude of the response generated. We therefore propose that RpoE1 is a secondary, species-specific regulator of SOE gene expression rather than a general, conserved regulatory circuit. Sulfite produced by major dissimilatory processes appeared to be the trigger for SOE gene expression in both species, as we were unable to find evidence for an increase of SOE activity in stationary growth phase. The basic regulation of bacterial sulfite oxidation by cognate ECF sigma factors is likely to be applicable to three groups of alpha and beta-Proteobacteria in which we identified similar SOE operon structures

Etude biochimique, structurale et physiologique d enzymes lipolytiques chez les Mycobactéries

Les enzymes lipolytiques chez Mycobacterium tuberculosis, l'agent étiologique de la tuberculose, restent peu étudiées malgré leur probable implication dans des fonctions physiologiques essentielles chez ce bacille. La mise à disposition de la séquence du génome de M. tuberculosis a permis d'identifier les gènes codant ces enzymes chez ce microorganisme. Mon travail de thèse a consisté, dans un premier temps, en la caractérisation biochimique, structurale et fonctionnelle d'une enzyme lipolytique potentielle, la Rv0183, chez M. tuberculosis. Nous avons démontré que la Rv0183 est une monoacylglycérol lipase localisée non seulement dans l'enveloppe mycobactérienne mais aussi dans le milieu de culture. Le rôle physiologique de la Rv0183 a été extrapolé grâce à l'étude de son homologue chez M. smegmatis, la MSMEG_0220. Cette protéine, en plus de son implication dans l'hydrolyse de lipides exogènes, semble jouer un rôle structural au niveau de l'enveloppe mycobactérienne. Dans un deuxième temps, nous nous sommes intéressés à la caractérisation de deux autres protéines secrétées par M. tuberculosis apparentées à la famille des cutinases : la Rv1984c et la Rv3452. Bien qu'elles présentent une identité de séquence de 50%, l'étude des propriétés cinétiques de ces protéines a montré une divergence dans leur spécificité de substrat. Si la Rv1984c pourrait être définie comme une lipase, la Rv3452 est une phospholipase de type A2. Etant secrétées par M. tuberculosis, une évaluation de la cytotoxicité de ces protéines a montré que contrairement à la Rv1984c, la Rv3452 était capable d'induire la lyse de macrophages suggérant une implication de cette protéine dans la virulence de M. tuberculosis. Un dernier aspect de mon travail a été d'étudier la lipolyse in situ chez M. smegmatis. En utilisant la microscopie de florescence en temps réel, nous avons suivi, l'hydrolyse des réserves intracytoplasmiques chez ce bacille placé en milieu appauvri. Ce phénomène a été bloqué par la la tétrahydrolipstatine confirmant l'implication d'enzymes lipolytiquesLipolytic enzymes in Mycobacterium tuberculosis, the etiologic agents of tuberculosis, remain poorly studied despite their probable involvement in bacteria cell life. Genomic DNA from M. tuberculosis analysis has permitted the identification of genes encoding lipolytic enzymes. My thesis work was consisted, on the one hand, of biochemical, structural and physiological characterization of Rv0183, a putative lipolytic enzyme from M. tuberculosis. We have demonstrated that Rv0183 is a monoacylglycerol lipase which is located not only in the cell envelope but also in the culture medium filtrate. The physiological function of Rv0183 was assessed by studying its ortholog in M. smegmatis, the MSMEG_0220. It has been shown that this protein was involved not only in the exogenous lipid degradation but also in the cell envelop architecture. On the other hand, we have investigated on the biochemical characterization of two secreted proteins of M. tuberculosis belonging to the cutinase family, Rv1984c and Rv3452. Despite 50% identity in their amino acid sequence, these enzymes show distinct substrate specificities. Rv1984c hydrolyses carboxyl esters and monoacylglycerols whereas Rv3452 behaves as a phospholipase A2 able to induce macrophage lysis suggesting an implication of this enzyme in M. tuberculosis virulence. Finally, we have studied the in situ lipolysis in M. smegmatis. Thanks to time-lapse fluorescence microscopy, we have monitored the intracytoplasmic lipids hydrolysis in this bacterium incubated under nutrient starvation. Lipid degradation was inhibited by tetrahydrolipstatin confirming the involvement of lipolytic enzymes in this processAIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Sulfur compound oxidation and carbon co-assimilation in the haloalkaliphilic sulfur oxidizers Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum

Alkaliphilic sulfur-oxidizing bacteria are highly abundant in naturally occurring soda lakes, where they are found both in surface waters and sediments. Here we studied oxidation of sulfide and thiosulfate in batch cultures of Thioalkalimicrobium aerophilum and Thioalkalivibrio versutus, two species that represent different metabolic types, as indicated by the absence or presence of sulfur production during growth, respectively. With thiosulfate, both species showed the expected sulfur oxidation patterns; however, during growth on sulfide, both T. aerophilum and T. versutus produced sulfur as an intermediate. While T. aerophilum likely uses a Sox-type sulfur oxidation pathway, T. versutus appeared to use a combination of some Sox proteins with heterodisulfide reductase complexes, which is supported by gene expression data. Interestingly, intermediate sulfur production by T. versutus occurred when the sulfur source in the medium had been nearly exhausted, which is unlike what has been described for the well-studied Dsr/Sox pathway in phototrophic sulfur bacteria. Inclusion of some carbon sources (acetate, propionate, fructose) slightly enhanced growth of T. versutus and T. aerophilum in batch cultures, suggesting that carbon co-assimilation may be occurring. Our results indicate that sulfur oxidation processes in alkaliphilic sulfur oxidizers are more complex than previously assumed, and that the enzymes involved warrant further study

Nitroxide functionalized antibiotics are promising eradication agents against Staphylococcus aureus biofilms

Treatment of biofilm-related Staphylococcus aureus infections represents an important medical challenge worldwide, as biofilms, even those involving drug-susceptible S. aureus strains, are highly refractory to conventional antibiotic therapy. Nitroxides were recently shown to induce the dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial biofilms remains unknown. Here, we demonstrate that the biofilm dispersal activity of nitroxides extends to S. aureus, a clinically important Gram-positive pathogen. Coadministration of the nitroxide CTEMPO (4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl) with ciprofloxacin significantly improved the biofilm eradication activity of the antibiotic against S. aureus. Moreover, covalently linking the nitroxide to the antibiotic moiety further reduced the ciprofloxacin minimal biofilm eradication concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids could penetrate S. aureus biofilms and enter cells localized at the surface and base of the biofilm structure. No toxicity to human cells was observed for the nitroxide CTEMPO or the nitroxide-antibiotic hybrids. Taken together, our results show that nitroxides can mediate the dispersal of Gram-positive biofilms and that dual-acting biofilm eradication antibiotics may provide broad-spectrum therapies for the treatment of biofilm-related infections.</p

Profluorescent fluoroquinolone-nitroxides for investigating antibiotic-bacterial interactions

Fluorescent probes are widely used for imaging and measuring dynamic processes in living cells. Fluorescent antibiotics are valuable tools for examining antibiotic-bacterial interactions, antimicrobial resistance and elucidating antibiotic modes of action. Profluorescent nitroxides are ‘switch on’ fluorescent probes used to visualize and monitor intracellular free radical and redox processes in biological systems. Here, we have combined the inherent fluorescent and antimicrobial properties of the fluoroquinolone core structure with the fluorescence suppression capabilities of a nitroxide to produce the first example of a profluorescent fluoroquinolone-nitroxide probe. Fluoroquinolone-nitroxide (FN) 14 exhibited significant suppression of fluorescence (36-fold), which could be restored via radical trapping (fluoroquinolone-methoxyamine 17) or reduction to the corresponding hydroxylamine 20. Importantly, FN 14 was able to enter both Gram-positive and Gram-negative bacterial cells, emitted a measurable fluorescence signal upon cell entry (switch on), and retained antibacterial activity. In conclusion, profluorescent nitroxide antibiotics offer a new powerful tool for visualizing antibiotic-bacterial interactions and researching intracellular chemical processes.</p

Draft genome sequences of three nontypeable strains of Haemophilus influenzae, C188, R535, and 1200, isolated from different types of disease

Nontypeable is a persistent human respiratory pathogen known to be involved in a range of acute and chronic respiratory diseases. Here, we report the genome sequences of three strains isolated from sputum, otitis media, and blood. Comparative analyses revealed significant differences in the gene contents including the presence of genes mediating antibiotic resistance

Eradicating uropathogenic Escherichia coli biofilms with a ciprofloxacin-dinitroxide conjugate

Urinary tract infections (UTIs) are amongst the most common and prevalent infectious diseases worldwide, with uropathogenic Escherichia coli (UPEC) reported as the main causative pathogen. Fluoroquinolone antibiotics are commonly used to treat UTIs but for infections involving UPEC biofilms, which are commonly associated with catheter use and recurrent episodes, ciprofloxacin is often ineffective. Here we report the development of a ciprofloxacin–dinitroxide (CDN) conjugate with potent UPEC biofilm-eradication activity. CDN 11 exhibited a 2-fold increase in potency over the parent antibiotic ciprofloxacin against UPEC biofilms. Moreover, CDN 11 resulted in almost complete UPEC biofilm cell eradication (99.7%) at concentrations as low as 12.5 μM, and significantly potentiated ciprofloxacin's biofilm-eradication activity against UPEC upon co-administration. The biofilm-eradication activity of CDN 11 highlights the potential of nitroxide functionalized antibiotics as a promising strategy for the treatment of biofilm-related UTIs

A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction▿ †

MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids

A high-throughput cell-based assay pipeline for the preclinical development of bacterial DsbA inhibitors as antivirulence therapeutics

Antibiotics are failing fast, and the development pipeline remains alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which inhibit bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors, previously identified by biophysical and biochemical assays. Inhibitors of DsbA block oxidative protein folding required for virulence factor folding in pathogens. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that the combination of a cell-based sulfotransferase assay and a motility assay (both DsbA reporter assays), modified for a higher throughput format, can provide a robust and target-specific platform for the identification and evaluation of DsbA inhibitors