Ana B. Gomez

IS6110 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.

Interactions of Attenuated Mycobacterium tuberculosis phoP Mutant with Human Macrophages

Background Mycobacterium tuberculosis phoP mutant SO2 derived from a clinical isolate was shown to be attenuated in mouse bone marrow-derived macrophages and in vivo mouse infection model and has demonstrated a high potential as attenuated vaccine candidate against tuberculosis. Methodology/Principal Findings In this study, we analyze the adhesion and the intracellular growth and trafficking of SO2 in human macrophages. Our results indicate an enhanced adhesion to phagocitic cells and impaired intracellular replication of SO2 in both monocyte-derived macrophages and human cell line THP-1 in comparison with the wild type strain, consistent with murine model. Intracellular trafficking analysis in human THP-1 cells suggest that attenuation of SO2 within macrophages could be due to an impaired ability to block phagosome-lysosome fusion compared with the parental M. tuberculosis strain. No differences were found between SO2 and the wild-type strains in the release and mycobacterial susceptibility to nitric oxide (NO) produced by infected macrophages. Conclusions/Significance SO2 has enhanced ability to bind human macrophages and differs in intracellular trafficking as to wild-type M. tuberculosis. The altered lipid profile expression of the phoP mutant SO2 and its inability to secrete ESAT-6 is discussed.

Mapping of IS6110 insertion sites in Mycobacterium bovis isolates in relation to adaptation from the animal to human host

The physiological role and impact of IS6110 insertions on the biology of Mycobacterium tuberculosis complex is not well understood. Insertion of IS6110 in coding regions can cause loss of gene activity, while homologous recombination between two copies of IS6110 can result in the deletion of genes or in rearrangement of genomic regions involved. In addition to these genomic changes, IS6110 can also activate flanking genes through acting as a mobile promoter. In order to determine the possible role of IS6110 transposition in the adaptation to humans, we selected Mycobacterium bovis isolates from endogenous reactivation cases in elderly people in The Netherlands. The human isolates contained higher number of IS6110 copies in comparison to the bovine M. bovis strains. These additional integration sites of IS6110 were sequenced and analyzed. From 12 of such IS6110 insertion sites, 6 loci were located in the intergenic regions, and 6 other occurred within coding regions. IS6110 was inserted in a position where it might serve as a promoter in two cases. We conclude that IS6110 transpositions in M. bovis may be a driving force in the adaptation from the animal to the human host.

Intracellular replication of attenuated Mycobacterium tuberculosis phoP mutant in the absence of host cell cytotoxicity.

Intracellular pathogen Mycobacterium tuberculosis survives and replicates in macrophages but limited information is available on its replication into non-phagocytic cells. Here we study the role of the M. tuberculosis virulence gene phoP in the intracellular growth with rat and human lung fibroblasts. In contrast to macrophages, attenuated M. tuberculosis phoP mutant was able to multiply intracellularly in fibroblasts at the same level as the virulent M. tuberculosis. However, when M. tuberculosis virulence was studied using human foetal lung fibroblasts, MRC-5 cell line, the virulent strain caused a significant damage in cells compared with attenuated strains BCG and M. tuberculosis phoP mutant. We analysed the effect of cytoskeleton inhibitors in NRK-49F fibroblasts. M. tuberculosis invasion was not inhibited, suggesting that mycobacterial uptake was microtubule and microfilament independent. Our results suggest that PhoP in M. tuberculosis does not regulate intracellular replication in fibroblasts, contrary to what happens in macrophages. The ability of M. tuberculosis phoP mutant to replicate within non-phagocytic cells, such as fibroblasts, without causing damage, could be a potential advantage for a live attenuated vaccine against tuberculosis.

Reactogenicity to major tuberculosis antigens absent in BCG is linked to improved protection against Mycobacterium tuberculosis

MTBVAC is a live-attenuated Mycobacterium tuberculosis vaccine, currently under clinical development, that contains the major antigens ESAT6 and CFP10. These antigens are absent from the current tuberculosis vaccine, BCG. Here we compare the protection induced by BCG and MTBVAC in several mouse strains that naturally express different MHC haplotypes differentially recognizing ESAT6 and CFP10. MTBVAC induces improved protection in C3H mice, the only of the three tested strains reactive to both ESAT6 and CFP10. Deletion of both antigens in MTBVAC reduces its efficacy to BCG levels, supporting a link between greater efficacy and CFP10- and ESAT6-specific reactogenicity. In addition, MTBVAC (but not BCG) triggers a specific response in human vaccinees against ESAT6 and CFP10. Our results warrant further exploration of this response as potential biomarker of protection in MTBVAC clinical trials.

Microscopic mild focal cortical dysplasia in temporal lobe dual pathology: An electrocorticography study

BACKGROUND Associations between electrophysiological and histological findings might provide an insight into the epileptogenicity of mild focal cortical dysplasia (FCD) in patients with temporal lobe epilepsy (TLE) and a dual pathology. SUBJECTS AND METHODS A total of 22 patients with pharmacoresistant TLE were included in the study, 16 of them with histologically confirmed hippocampal sclerosis (HS) associated with neocortical temporal mild Palmini Type-I FCD subtypes and 6 with HS. Intraoperative electrocorticography (ECoG) recordings were analysed for epileptiform discharge frequency and morphology. Associations between histological, and electrocorticography pattern findings in these patients were analysed. Electroclinical outcomes in these patients were also evaluated. RESULTS Neocortical areas with mild Palmini Type-I FCD showed a significantly higher spike frequency (SF) recorded in the inferior temporal gyrus than those neocortical areas in patients with HS. There was a tendency to higher spike frequency and lower amplitude in neocortical areas with histopathologic subtype IB FCD in relation with IA during intraoperative ECoG. Post-SF excision and amplitude were significantly lower during neocortical post-excision intraoperative ECoG than during neocortical pre-excision recording. There was no difference found in the clinical outcome between patients with and without FCD. CONCLUSIONS Intraoperative electrocorticographic interictal spike frequency recorded in the neocortical inferior temporal gyrus may help to characterize the histopathologic subtypes of mild Palmini Type-I FCD in patients with temporal lobe epilepsy (TLE) and a dual pathology. Our data support the epileptogenicity of neocortical mild FCD in TLE and assessments of ECoG patterns are relevant to determine the extent of the resection in these patients which can influence the electroclinical outcome.

Hyper-attenuated MTBVAC erp mutant protects against tuberculosis in mice

Safety of individuals at risk of immune suppression is an important concern for live vaccines. The new-generation tuberculosis vaccine candidate MTBVAC, a genetically engineered doubly attenuated Mycobacterium tuberculosis mutant with deletions in phoP and fadD26 virulence genes has demonstrated comparable safety in different relevant animal models and superior protection in mice as compared to the only currently licensed tuberculosis vaccine Mycobacterium bovis BCG. Here we describe the construction of a highly attenuated MTBVAC-based live vaccine by an additional gene inactivation generated in erp of MTBVAC. The gene product of erp is an exported repeated protein (Erp), a virulence factor described to be involved in intracellular replication of M. tuberculosis. The resultant strain, MTBVAC erp(-), was tested in severe combined immunodeficiency (SCID) mouse model showing to be severely attenuated when compared to BCG and MTBVAC. Experiments conducted in immunocompetent mice revealed that the hyper-attenuated profile observed with MTBVAC erp(-) strain did not compromise its protective efficacy profile in comparison with BCG. These results postulate MTBVAC erp(-) as a potential tuberculosis vaccine candidate for use in high-risk populations of immune suppression (e.g., due to HIV infection), where the use of BCG is not recommended.

Therapeutic efficacy of the live-attenuated Mycobacterium tuberculosis vaccine, MTBVAC, in a preclinical model of bladder cancer

&NA; Intravesical instillation of bacillus Calmette‐Guérin (BCG) has been a first‐line therapy for non–muscle‐invasive bladder cancer for the last 4 decades. However, this treatment causes serious adverse events in a significant number of patients and a substantial percentage of recurrence episodes. MTBVAC is a live‐attenuated vaccine derived from a Mycobacterium tuberculosis clinical isolate and is currently under evaluation in clinical trials to replace BCG as a tuberculosis vaccine. Here, we describe for the first time the potential of MTBVAC as a bladder cancer therapy in vitro and in vivo in a preclinical model. MTBVAC colonized human bladder tumor cells to a much greater extent than BCG via a mechanism mediated by macropinocytosis and induced cell growth inhibition after internalization. In vivo testing in an orthotopic murine model of bladder cancer demonstrated a higher antitumor effect of MTBVAC in experimental conditions in which BCG did not work. Our data encourage further studies to support the possible application of MTBVAC as a new immunotherapeutic agent for bladder cancer.