IS 6110 Mediates Increased Transcription of the phoP Virulence Gene in a Multidrug-Resistant Clinical Isolate Responsible for Tuberculosis Outbreaks

Abstract

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...

BCG Vaccines: Their mechanisms of attenuation and impact on safety and protective efficacy

“…66 H37Ra is also defective in PDIMs, but this phenotype is not caused by the phoP mutation. 63 Collectively, these studies have demonstrated that the PhoP-PhoR system, particularly PhoP, plays an essential role in M. tb virulence. 62,65,66,69 Considering its prominent role in virulence, it may not be surprising that BCG strains exhibit a number of genetic polymorphisms in the phoP-phoR locus.…”

“…69 In M. tb, defective biosynthesis of trehalose-containing lipids does not explain the attenuated phenotype of the phoP mutant, but impaired ESX-1 secretion might. 63 In the avirulent lab strain, H37Ra, attenuation is partially explained by a single point mutation (S219L) in the DNA binding region of PhoP. 66 H37Ra is also defective in PDIMs, but this phenotype is not caused by the phoP mutation.…”

“…61 The PhoR protein is a transmembrane histidine kinase that transmits signals from the environment. Autophosphorylation of PhoR is followed by transfer of the phosphoryl group to PhoP, a response regulator that mediates expression of multiple genes, 62 including genes for the biosynthesis of trehalose-containing cell wall lipids [63][64][65] and ESX-1 secretion. 66 A role for phoP in virulence of the M. tb complex was first suggested during a severe nosocomial outbreak of multidrug resistant TB in humans in Spain.…”

Physiological impact of transposable elements encoding DDE transposases in the environmental adaptation of Streptococcus agalactiae

“…Transposable elements can also act by modifying the expression of neighbouring genes at the integration site, or by promoting huge genomic rearrangements when they are substrates for homologous recombination (Casacuberta & González, 2013;Mahillon & Chandler, 1998;Maruyama et al, 2009). Some well-described bacterial examples are the activation of the cryptic bgl and cel operons required for utilization of b-glucoside sugars in Escherichia coli by disruption of silencer elements around the promoter region by IS1, IS2 or IS5 transposition; IS1 insertion into the mgl locus improving the glucose transport and fitness of E. coli; ISS12 integration into the srpS gene increasing the resistance of Pseudomonas putida to toluene by derepressing the genes encoding an inducible solvent resistance pump; or IS6110 controlling transcription of Mycobacterium tuberculosis genes (including the phoP virulence gene) downstream of its integration site through an outward-directed promoter located at its 39 end (Hall, 1998;Notley-McRobb & Ferenci, 1999;Parker & Hall, 1990;Safi et al, 2004;Soto et al, 2004;Wery et al, 2001).…”

Epidemiología molecular de la tuberculosis: métodos y aplicaciones

“…Another example of molecular mechanisms possibly involved in virulence of drug resistant strains is the regulation of genes mediated by IS6110. This insertion sequence appears to be associated with the upregulation of genes implicated in virulence of Beijing strains and other clinical strains that have caused MDR-TB outbreaks (101,102). Further studies with this family of strains will reveal the molecular mechanisms involved in virulence, if in fact these strains are more virulent than others, and their impact on the worldwide tuberculosis epidemic.…”