Two-component regulatory signal transduction systems are important elements of the adaptative response of prokaryotes to a variety of environmental stimuli. Disruption of PhoP-PhoR in Mycobacterium tuberculosisdramatically attenuates virulence, implying that this system directly and/or indirectly coordinates the expression of important virulence factors whose identity remains to be established. Interestingly, in knockingout the PhoP-PhoR two-component system in M. tuberculosis Mt103, dramatic changes in the colonial morphology, cording properties, and reactivity of the mutant strain to the basic dye neutral red, all intrinsic properties of tubercle bacilli known to correlate with virulence, were noted. Because deficiencies in the ability of the mutant to form serpentine cords and stain with the dye are likely the results of alterations of its cell envelope composition, we undertook to analyze the lipid content of phoP and phoP-phoR mutants constructed in two different strains of M. tuberculosis. Our results indicate that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. Evidence is also provided that PhoP but not PhoR is required for the production of these lipids. This work represents an important step toward the functional characterization of PhoP-PhoR and the understanding of complex lipid synthesis in M. tuberculosis.Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is one of the leading causes of mortality due to a single infectious agent (1). In the tubercle bacillus as in other prokaryotes, two-component signal transduction systems are important elements of the adaptative response to a variety of stimuli (2). So far, of the 11 paired two-component systems, 5 unpaired response regulators and 2 unpaired protein sensors that M. tuberculosis possesses, the two-component system PhoP-PhoR is the one whose disruption was shown to affect the most dramatically the ability of M. tuberculosis to replicate in cellular and animal models (3). Interestingly, PhoP shows high similarity to the PhoP response regulator of Salmonella enterica serovar typhimurium, which senses Mg 2ϩ starvation and controls the expression of at least 40 genes, among which some encoding important virulence determinants (4). Further supporting the concept that PhoP is important for virulence and transmissibility of tubercle bacilli, a multidrug-resistant strain of Mycobacterium bovis (strain B) responsible for large tuberculosis outbreaks in Spain was found to carry an IS6110 insertion in the promoter region of phoP causing a strong up-regulation of the expression of this gene (5). To date, the stimuli sensed by the sensor histidine kinase PhoR and the genes controlled by the DNA-binding response regulator PhoP are not known. The identification of the environmental signals regulating PhoP-PhoR and the characterization of t...

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Drug resistance in Mycobacterium tuberculosis complex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR) M. tuberculosis complex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of M. bovis. Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS6110. Here, we mapped and sequenced the regions flanking the two copies of IS6110 in the B strain. Ligation-mediated PCR showed that one of these IS6110 copies is located within the promoter region of phoP, a transcriptional regulator that is essential for M. tuberculosis virulence. We used PCR to screen 219 MDR M. tuberculosis complex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS6110 in the phoP promoter. To determine whether IS6110 affects phoP promoter activity in the B strain, we individually cloned the phoP gene and its promoter region (including IS6110 from the B strain and the equivalent region from M. tuberculosis without IS6110 as a control) into a mycobacterial replicative plasmid and transformed M. smegmatis with the resulting plasmid. Primer extension analysis showed that phoP transcription was strongly upregulated when the promoter region contained IS6110, as in the case of the B strain.Tuberculosis (TB) is currently one of the leading causes of mortality throughout the world (8,25,27). The human immunodeficiency virus-AIDS pandemic, the deterioration of public health systems in developing countries, and the emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains have further contributed to the spread of TB. Knowledge of the molecular mechanisms involved in the bacillus-host cell interaction is essential for developing adequate strategies for TB control. Recent advances in the genetic manipulation of mycobacteria (2, 29) combined with the publication of the complete M. tuberculosis genome sequence (6) have made it possible to study the contribution of individual genes to M. tuberculosis virulence (5, 7). However, little is known about the regulatory and expression mechanisms that determine the virulence of clinical isolates of M. tuberculosis.Insertion sequence (IS) 6110 has been extensively used for molecular typing of M. tuberculosis strains. Restriction fragment length polymorphism (RFLP) analysis using IS6110 as a probe is currently the most common molecular method used to type M. tuberculosis complex strains. However, the physiological role and impact of specific IS6110 insertions on the biology of bacilli are not well known. IS6110 fingerprinting studies have demonstrated heterogeneity between virulence and transm...

A strong antimicrobial activity against Escherichia coli of Cu‐BTC metal‐organic frameworks immobilized over cellulosic fibers is hereby reported. The in situ synthesis of Cu‐BTC metal‐organic frameworks, aka MOF‐199 or HKUST‐1, onto cellulosic substrates was carried out by exposing carboxymethylated cellulosic substrates to Cu(OAC)2, 1,3,5‐benzenetricarboxylic acid and triethylamine solutions following a very specific order. Using an in vitro model, in accordance to ASTM E2149‐13a, we observed that the cellulose‐MOF system was able to completely eliminate the growth of E. coli on agar plates and liquid cultures. The antibacterial activity of the comprising components of MOF‐199 and the cellulosic substrate was also evaluated and determined to be negligible. Since the method used to synthesize MOF‐199 crystals provides a strong bond between the crystals and the cellulosic substrates, the crystals not detach from the anionic cellulosic fibers allowing the modified textile to be washed and reused hence opening a new avenue to fabricate antibacterial clinical fabrics. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40815.

The attenuated Mycobacterium tuberculosis H37Ra strain is an isogenic counterpart of the virulent paradigm strain H37Rv. Recently, a link between a point mutation in the PhoP transcriptional regulator and avirulence of H37Ra was established. Remarkably, a previous study demonstrated negative autoregulation of the phoP gene in H37Ra. These findings led us to study the transcriptional autoregulation of PhoP in the virulent H37Rv strain. In contrast to the negative autoregulation of PhoP previously published for H37Ra, our experiments using a phoP promoter-lacZ fusion showed that PhoP is positively autoregulated in both H37Rv and H37Ra compared with an H37Rv phoP deletion mutant constructed in this study. Using quantitative reverse transcription-PCR (RT-PCR) analysis, we showed that the phoP gene is transcribed at similar levels in H37Rv and H37Ra. Gel mobility shift and DNase I footprinting assays allowed us to identify the precise binding region of PhoP from H37Rv to the phoP promoter. We also carried out RT-PCR studies to demonstrate that phoP is transcribed together with the adjacent gene phoR, which codes for the cognate histidine kinase of the phoPR two-component system. In addition, quantitative RT-PCR studies showed that phoR is independently transcribed from a promoter possibly regulated by PhoP. Finally, we discuss the possible role in virulence of a single point mutation found in the phoP gene from the attenuated H37Ra strain but not in virulent members of the M. tuberculosis complex.