BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine.

Recent publications have explored intranasal (i.n.) adenovirus-based (Ad) vaccines as an effective strategy for SARS-CoV-2 in pre-clinical models. However, the effects of prior immunizations and infections have yet to be considered. Here, we investigate the immunomodulatory effects of Mycobacterium bovis BCG pre-immunization followed by vaccination with an S-protein-expressing i.n. Ad, termed Ad(Spike). While i.n. Ad(Spike) retains some protective effect after 6 months, a single administration of BCG-Danish prior to Ad(Spike) potentiates its ability to control viral replication of the B.1.351 SARS-CoV-2 variant within the respiratory tract. Though BCG-Danish did not affect Ad(Spike)-generated humoral immunity, it promoted the generation of cytotoxic/Th1 responses over suppressive FoxP3+ TREG cells in the lungs of infected mice. Thus, this vaccination strategy may prove useful in limiting future pandemics by potentiating the long-term efficacy of mucosal vaccines within the context of the widely distributed BCG vaccine.

[Wellens syndrome: A high-risk electrocardiographic pattern]. / Síndrome de Wellens: un patrón electrocardiográfico de alto riesgo.

We present the case of a 40-year-old male without cardiovascular risk factors, who was admitted to our hospital due to chest pain. The electrocardiogram showed a typical pattern, already described, known as Wellens syndrome type 1 or A, characterized by ST segment elevation <1 mm and biphasic T wave in lead V3. Was diagnosed of acute myocardial infarction without ST elevation, TIMI 3 and GRACE score 66 points. This electrocardiographic pattern is of high risk, beyond the stratifying scores, since it correlates with severe injury to the anterior descending artery at the proximal level and requires percutaneous intervention without delay. The coronary angiography revealed 3 severe lesions: the proximal and middle thirds of the left anterior descending artery and the first diagonal. We highlight the validity of this electrocardiographic pattern, described more than 40 years ago, to identify and stratify patients with acute coronary syndrome.

Se presenta el caso de un varón de 40 años sin antecedentes cardiovasculares, que ingresó a nuestro centro por dolor precordial. En el electrocardiograma se evidenció un patrón típico, ya descrito, conocido como síndrome de Wellens tipo 1 o A, caracterizado por supradesnivel del ST < 1 mm y onda T bifásica en la derivación V3. Se diagnosticó infarto agudo de miocardio sin elevación del ST, TIMI 3 y GRACE score 66 puntos. Este patrón electrocardiográfico es de alto riesgo, más allá de los puntajes estratificadores, dado que se correlaciona con lesión grave en la arteria descendente anterior a nivel proximal y requiere intervención percutánea sin demoras. En la coronariografía se evidenciaron 3 lesiones graves: tercio proximal y medio de la arteria descendente anterior y primera diagonal. Destacamos la vigencia de este patrón electrocardiográfico, descrito hace más de 40 años, para identificar y estratificar a pacientes con síndrome coronario agudo.

Síndrome de Wellens: un patrón electrocardiográfico de alto riesgo / Wellens syndrome: A high-risk electrocardiographic pattern

Resumen Se presenta el caso de un varón de 40 años sin antecedentes cardiovasculares, que ingresó a nuestro centro por dolor precordial. En el electrocardiograma se evidenció un patrón típico, ya des crito, conocido como síndrome de Wellens tipo 1 o A, caracterizado por supradesnivel del ST < 1 mm y onda T bifásica en la derivación V3. Se diagnosticó infarto agudo de miocardio sin elevación del ST, TIMI 3 y GRACE score 66 puntos. Este patrón electrocardiográfico es de alto riesgo, más allá de los puntajes estratificadores, dado que se correlaciona con lesión grave en la arteria descendente an terior a nivel proximal y requiere intervención percutánea sin demoras. En la coronariografía se eviden ciaron 3 lesiones graves: tercio proximal y medio de la arteria descendente anterior y primera diagonal. Destacamos la vigencia de este patrón electrocardiográfico, descrito hace más de 40 años, para identificar y estratificar a pacientes con síndrome coronario agudo.

Abstract We present the case of a 40-year-old male without cardiovascular risk factors, who was admit ted to our hospital due to chest pain. The electrocardiogram showed a typical pattern, already described, known as Wellens syndrome type 1 or A, characterized by ST segment elevation <1 mm and biphasic T wave in lead V3. Was diagnosed of acute myocardial infarction without ST elevation, TIMI 3 and GRACE score 66 points. This electrocardiographic pattern is of high risk, beyond the stratifying scores, since it correlates with severe injury to the anterior descending artery at the proximal level and requires percutaneous interven tion without delay. The coronary angiography revealed 3 severe lesions: the proximal and middle thirds of the left anterior descending artery and the first diagonal. We highlight the validity of this electrocardiographic pattern, described more than 40 years ago, to identify and stratify patients with acute coronary syndrome.

Experimental Confirmation that an Uncommon rrs Gene Mutation (g878a) of Mycobacterium tuberculosis Confers Resistance to Streptomycin.

The effective treatment of patients diagnosed with drug-resistant tuberculosis is highly dependent on the ability to rapidly and accurately determine the antibiotic susceptibility profile of the Mycobacterium tuberculosis isolate(s) involved. Thus, as more clinical microbiology laboratories advance toward the use of DNA sequence-based diagnostics, it is imperative that their predictive functions extend beyond the well-known resistance mutations in order to also encompass as many of the lower-frequency mutations as possible. However, in most cases, fundamental experimental proof that links these uncommon mutations with phenotypic resistance is lacking. One such example is the g878a polymorphism within the rrs 16S rRNA gene. We, and others, have identified this mutation within a small number of drug-resistant isolates, although a consensus regarding exactly which aminoglycoside antibiotic(s) it confers resistance to has not previously been reached. Here, we have employed oligonucleotide-mediated recombineering to introduce the g878a polymorphism into the rrs gene of Mycobacterium bovis BCG, a close relative of M. tuberculosis, and demonstrate that it confers low-level resistance to streptomycin alone. It does not confer cross-resistance to amikacin, capreomycin, or kanamycin. We also demonstrate that the rrsg878a mutation exerts a substantial fitness defect in vitro that may at least in part explain why clinical isolates bearing this mutation appear to be quite rare. Overall, this study provides clarity to the phenotype attributable to the rrsg878a mutation and is relevant to the future implementation of genomics-based diagnostics as well as the clinical management of patients in whom this particular polymorphism is encountered.

[Takotsubo syndrome and multivessel coronary disease]. / Síndrome de Takotsubo y enfermedad multivaso.

Takotsubo syndrome is a generally reversible cardiomyopathy often related to a stressor trigger, either physical or emotional. It is estimated that this entity represents between 1 and 6% of the cases of suspected acute coronary syndrome without ST segment elevation in women. Coexistence with coronary artery disease has been a matter of long controversy. On this matter, we present the clinical case of a 79-year-old hypertensive and dyslipidemic female with smocking history, who was referred to our institution presenting an episode of chest pain with anginal characteristics. During the conducted interrogation, she reported having been overpassing a stressfull moment in her private life. The electrocardiogram showed deep and diffuse negative T waves with prolonged QT interval. Laboratory findings revealed dissociation of biomarkers (troponin/ProBNP), and the transthoracic Doppler echocardiogram showed left ventricular dysfunction with apical ballooning. Given the differential diagnoses of acute coronary syndrome and TakoTsubo syndrome, a coronary angiography was performed, which revealed multiple epicardial coronary disease. However, due to the strong suspicion of stress cardiomyopathy, a cardiac magnetic resonance imaging with gadolinium was performed, which showed an improvement in the ejection fraction prior to revascularization with the presence of myocardial edema and absence of late gadolinium enhancement. The aforementioned characteristics led to the diagnosis of Takotsubo syndrome. The final decision of revascularization was based on the fact that the patient's principal complaint was typical anginal symptoms.

El síndrome de Takotsubo es una miocardiopatía generalmente reversible y con frecuencia relacionada a un desencadenante estresor ya sea físico o emocional. Se estima que esta entidad representa entre el 1 y 6% de los cuadros de sospecha de síndrome coronario agudo sin elevación del segmento ST en mujeres. La coexistencia con enfermedad coronaria ha sido objetivo de debate durante mucho tiempo. Se presenta el caso clínico de una mujer de 79 años, hipertensa, dislipémica y ex tabaquista que consulta a la guardia por presentar un episodio de dolor precordial con características anginosas. En el interrogatorio refirió situación estresante en su entorno familiar los días previos. El electrocardiograma mostró ondas T negativas profundas y difusas con prolongación del intervalo QT. En el laboratorio se observó disociación de marcadores (troponina/ ProBNP). En el ecocardiograma Doppler transtorácico se evidenció deterioro de la función ventricular con balonamiento apical. Ante los diagnósticos diferenciales de síndrome coronario agudo y síndrome de Takotsubo se realizó una cinecoronariografía en la cual se evidenció compromiso de las tres arterias coronarias epicárdicas. Pese a esto, por la fuerte sospecha de miocardiopatía por estrés se realizó una resonancia magnética cardíaca con gadolinio, la cual demostró mejoría de fracción de eyección previa a la revascularización con presencia de edema miocárdico y sin realce tardío de gadolinio. Las características mencionadas condujeron al diagnóstico de síndrome de Takotsubo. La decisión de revascularización, estuvo fundamentada en el hecho de que la consulta fue motivada por síntomas anginosos típicos con el esfuerzo.

The Diverse Applications of Recombinant BCG-Based Vaccines to Target Infectious Diseases Other Than Tuberculosis: An Overview.

Live attenuated Bacillus Calmette-Guérin (BCG) is the world's most widely used vaccine which is mainly administered for its protection against tuberculosis (TB), particularly in young children. However, since its initial use over 100years ago, it has also proven to offer a level of protection against various other pathogens, as a consequence of its non-specific immune enhancing effects. Thus, over the past few decades, recombinant BCG (rBCG) technology has been used as a vector to create rBCG vaccines expressing heterologous antigens that elicit immunity against a range of bacterial, viral, and parasitic diseases. Our goal with this mini-review is to provide an up-to-date survey of the various techniques, approaches, and applications of rBCG-based vaccines for targeting infectious diseases other than TB.

Síndrome de Takotsubo y enfermedad multivaso / Takotsubo syndrome and multivessel coronary disease

Resumen El síndrome de Takotsubo es una miocardiopatía generalmente reversible y con frecuencia relacionada a un desencadenante estresor ya sea físico o emocional. Se estima que esta entidad representa entre el 1 y 6% de los cuadros de sospecha de síndrome coronario agudo sin elevación del segmento ST en mujeres. La coexistencia con enfermedad coronaria ha sido objetivo de debate durante mucho tiempo. Se pre senta el caso clínico de una mujer de 79 años, hipertensa, dislipémica y ex tabaquista que consulta a la guardia por presentar un episodio de dolor precordial con características anginosas. En el interrogatorio refirió situación estresante en su entorno familiar los días previos. El electrocardiograma mostró ondas T negativas profundas y difusas con prolongación del intervalo QT. En el laboratorio se observó disociación de marcadores (troponina/ ProBNP). En el ecocardiograma Doppler transtorácico se evidenció deterioro de la función ventricular con balo namiento apical. Ante los diagnósticos diferenciales de síndrome coronario agudo y síndrome de Takotsubo se realizó una cinecoronariografía en la cual se evidenció compromiso de las tres arterias coronarias epicárdicas. Pese a esto, por la fuerte sospecha de miocardiopatía por estrés se realizó una resonancia magnética cardíaca con gadolinio, la cual demostró mejoría de fracción de eyección previa a la revascularización con presencia de edema miocárdico y sin realce tardío de gadolinio. Las características mencionadas condujeron al diagnóstico de síndrome de Takotsubo. La decisión de revascularización, estuvo fundamentada en el hecho de que la consulta fue motivada por síntomas anginosos típicos con el esfuerzo.

Abstract Takotsubo syndrome is a generally reversible cardiomyopathy often related to a stressor trigger, either physical or emotional. It is esti mated that this entity represents between 1 and 6% of the cases of suspected acute coronary syndrome without ST segment elevation in women. Coexistence with coronary artery disease has been a matter of long controversy. On this matter, we present the clinical case of a 79-year-old hypertensive and dyslipidemic female with smocking history, who was referred to our institution presenting an episode of chest pain with anginal characteristics. During the conducted interrogation, she reported having been overpassing a stressfull moment in her private life. The electrocardiogram showed deep and diffuse negative T waves with prolonged QT interval. Laboratory findings revealed dissociation of biomarkers (troponin/ProBNP), and the transthoracic Doppler echocardiogram showed left ventricular dysfunction with apical ballooning. Given the differential diagnoses of acute coronary syndrome and TakoTsubo syndrome, a coronary angiography was performed, which revealed multiple epicardial coronary disease. However, due to the strong suspicion of stress cardiomyopathy, a cardiac magnetic resonance imaging with gadolinium was performed, which showed an improvement in the ejection fraction prior to revasculariza tion with the presence of myocardial edema and absence of late gadolinium enhancement. The aforementioned characteristics led to the diagnosis of Takotsubo syndrome. The final decision of revascularization was based on the fact that the patient's principal complaint was typical anginal symptoms.

Heterologous Production of 1-Tuberculosinyladenosine in Mycobacterium kansasii Models Pathoevolution towards the Transcellular Lifestyle of Mycobacterium tuberculosis.

Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24 h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and Ccr2-/- mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation.IMPORTANCE This work sheds light on the role of the lipid 1-tuberculosinyladenosine in the evolution of an environmental ancestor to M. tuberculosis On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen.

Quick Analysis of Organic Amendments via Portable X-ray Fluorescence Spectrometry.

The determination of heavy metals in soils and organic amendments, such as compost, manure, biofertilizer, and sludge, generally involves the digestion of samples with aqua regia, and the determination of those in the solution using various techniques. Portable X-ray fluorescence (PXRF) has many advantages in relation to traditional analytical techniques. However, PXRF determines the total elemental content and, until now, its use for the analysis of organic amendments has been limited. The objective of this work is the calibration of a PXRF instrument to determine the aqua regia-soluble elemental contents directly in solid samples of organic amendments. Our proposal will avoid the digestion step and the use of other laboratory techniques. Using a training set of samples, calibration functions were obtained that allow the determination of the aqua regia-soluble contents from the PXRF readings of total contents. The calibration functions (obtained by multiple linear regression) allowed the quantitative determination of the aqua regia-soluble contents of Fe, K, P, S, Zn, Cu, Pb, Sr, Cr, and Mn, as well as the organic matter content and a semi-quantitative assessment of Al, Ca, V, Ba, Ni, and As contents. The readings of Si, Fe, Al, Ca, K, or S were used as correction factors, indicating that the calibrations functions found are truly based on the chemical composition of the sample matrix. This study will allow a fast, cheap, and reliable field analysis of organic amendments and of other biomass-based materials.

Kidney-lung pathophysiological crosstalk: its characteristics and importance.

Crosstalk between the lung and the kidney is based on the similarities that these organs share. This is why different diseases that affect one organ can have repercussions on the other. Patients with acute kidney injury can present complications such as pulmonary edema and require mechanical ventilation in respiratory failure. This interaction occurs due to the increase in systemic immune mediators that cause inflammatory reactions, oxidative stress, and an increase in vascular permeability in the lung. With regard to lung-induced renal damage, the kidney can also be affected by chemical mediators, which are translocated into the bloodstream. Moreover, the kidneys are extremely sensitive to oxygen changes which can cause them to lose their autoregulation mechanism. In patients with acute lung injury (ALI), oxygen supply is decreased causing renal hypoxia. Besides, hypercapnia generated by ALI causes vasoconstriction in the renal vascular network and activation of the renal angiotensin aldosterone system. ALI not only can cause renal injury, but also worsening chronic obstructive pulmonary disease and obstructive sleep apnea. In conclusion, kidney-lung crosstalk is commonly present in certain pathological states, and knowing its characteristics is crucial for managing the complications which may arise from this vicious circle.

Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis.

The DosR regulon, a set of 48 genes normally expressed in Mycobacterium tuberculosis under conditions that inhibit aerobic respiration, is controlled via the DosR-DosS/DosT two-component system. While the regulon requires induction in most M. tuberculosis isolates, for members of the Beijing lineage, its expression is uncoupled from the need for signaling. In our attempts to understand the mechanistic basis for this uncoupling in the Beijing background, we previously reported the identification of two synonymous single-nucleotide polymorphisms (SNPs) within the adjacent Rv3134c gene. In the present study, we have interrogated the impact of these SNPs on dosR expression in wild-type strains, as well as a range of dosR-dosS-dosT mutants, for both Beijing and non-Beijing M. tuberculosis backgrounds. In this manner, we have unequivocally determined that the C601T dosR promoter SNP is the sole requirement for the dramatic shift in the pattern of DosR regulon expression seen in this globally important lineage. Interestingly, we also show that DosT is completely nonfunctional within these strains. Thus, a complex series of evolutionary steps has led to the present-day Beijing DosR phenotype that, in turn, potentially confers a fitness advantage in the face of some form of host-associated selective pressure. IMPORTANCE: Mycobacterium tuberculosis strains of the Beijing lineage have been described as being of enhanced virulence compared to other lineages, and in certain regions, they are associated with the dramatic spread of multidrug-resistant tuberculosis (TB). In terms of trying to understand the functional basis for these broad epidemiological phenomena, it is interesting that, in contrast to the other major lineages, the Beijing strains all constitutively overexpress members of the DosR regulon. Here, we identify the mutational events that led to the evolution of this unique phenotype. In addition, our work highlights the fact that important phenotypic differences exist between distinct M. tuberculosis lineages, with the potential to impact the efficacy of diagnosis, vaccination, and treatment programs.

Iron Acquisition in Mycobacterium avium subsp. paratuberculosis.

UNLABELLED: Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSP(P)15. We obtained a transposon (Tn) mutant with a disruption in the LSP(P)15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSP(P)15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen. IMPORTANCE: Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely related M. avium subspecies, mycobactin production and iron uptake are different in M. avium subsp. paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSP(P)15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer in M. avium subsp. paratuberculosis evolution.

Reemergence and amplification of tuberculosis in the Canadian arctic.

BACKGROUND: Between November 2011 and November 2012, a Canadian village of 933 persons had 50 culture-positive cases of tuberculosis, with 49 sharing the same genotype. METHODS: We performed Illumina-based whole-genome sequencing on Mycobacterium tuberculosis isolates from this village, during and before the outbreak. Phylogenetic trees were generated using the maximum likelihood method. RESULTS: Three distinct genotypes were identified. Strain I (n = 7) was isolated in 1991-1996. Strain II (n = 8) was isolated in 1996-2004. Strain III (n = 62) first appeared in 2007 and did not arise from strain I or II. Within strain III, there were 3 related but distinct clusters: IIIA, IIIB, and IIIC. Between 2007 and 2010, cluster IIIA predominated (11 of 22 vs 2 of 40; P < .001), whereas in 2011-2012 clusters IIIB (n = 18) and IIIC (n = 20) predominated over cluster IIIA (n = 11). Combined evolutionary and epidemiologic analysis of strain III cases revealed that the outbreak in 2011-2012 was the result of ≥6 temporally staggered events, spanning from 1 reactivation case to a point-source outbreak of 20 cases. CONCLUSIONS: After the disappearance of 2 strains of M. tuberculosis in this village, its reemergence in 2007 was followed by an epidemiologic amplification, affecting >5% of the population.

Origins of a 350-kilobase genomic duplication in Mycobacterium tuberculosis and its impact on virulence.

In the present study, we have investigated the evolution and impact on virulence of a 350-kb genomic duplication present in the most recently evolved members of the Mycobacterium tuberculosis East Asian lineage. In a mouse model of infection, comparing HN878 subclones HN878-27 (no duplication) and HN878-45 (with the 350-kb duplication) revealed that the latter is impaired for in vivo growth during the initial 3 weeks of infection. Furthermore, the median survival time of mice infected with isolate HN878-45 is significantly longer (77 days) than that of mice infected with HN878-27. Whole-genome sequencing of both isolates failed to reveal any mutational events other than the duplication that could account for such a substantial difference in virulence. Although we and others had previously speculated that the 350-kb duplication arose in response to some form of host-applied selective pressure (P. Domenech, G. S. Kolly, L. Leon-Solis, A. Fallow, M. B. Reed, J. Bacteriol. 192: 4562-4570, 2010, and B. Weiner, J. Gomez, T. C. Victor, R. M. Warren, A. Sloutsky, B. B. Plikaytis, J. E. Posey, P. D. van Helden, N. C. Gey van Pittius, M. Koehrsen, P. Sisk, C. Stolte, J. White, S. Gagneux, B. Birren, D. Hung, M. Murray, J. Galagan, PLoS One 7: e26038, 2012), here we show that this large chromosomal amplification event is very rapidly selected within standard in vitro broth cultures in a range of isolates. Indeed, subclones harboring the duplication were detectable after just five rounds of in vitro passage. In contrast, the duplication appears to be highly unstable in vivo and is negatively selected during the later stages of infection in mice. We believe that the rapid in vitro evolution of M. tuberculosis is an underappreciated aspect of its biology that is often ignored, despite the fact that it has the potential to confound the data and conclusions arising from comparative studies of isolates at both the genotypic and phenotypic levels.

N-glycolylated peptidoglycan contributes to the immunogenicity but not pathogenicity of Mycobacterium tuberculosis.

Mycobacteria produce an unusual, glycolylated form of muramyl dipeptide (MDP) that is more potent and efficacious at inducing NOD2-mediated host responses. We tested the importance of this modified form of MDP in Mycobacterium tuberculosis by disrupting the gene, namH, responsible for this modification. In vitro, the namH mutant did not produce N-glycolylated muropeptides, but there was no alteration in colony morphology, growth kinetics, cellular morphology, or mycolic acid profile. Ex vivo, the namH mutant survived and replicated normally in murine and human macrophages, yet induced diminished production of tumor necrosis factor α. In vivo, namH disruption did not affect the bacterial burden during infection of C57BL/6 mice or cellular recruitment to the lungs but modestly prolonged survival after infection in Rag1(-/-) mice. These results indicate that the modified MDP is an important contributor to the unusual immunogenicity of mycobacteria but has a limited role in the pathogenesis of M. tuberculosis infection.

Massive gene duplication event among clinical isolates of the Mycobacterium tuberculosis W/Beijing family.

As part of our effort to uncover the molecular basis for the phenotypic variation among clinical Mycobacterium tuberculosis isolates, we have previously reported that isolates belonging to the W/Beijing lineage constitutively overexpress the DosR-regulated transcriptional program. While generating dosR knockouts in two independent W/Beijing sublineages, we were surprised to discover that they possess two copies of dosR. This dosR amplification is part of a massive genomic duplication spanning 350 kb and encompassing >300 genes. In total, this equates to 8% of the genome being present as two copies. The presence of IS6110 elements at both ends of the region of duplication, and in the novel junction region, suggests that it arose through unequal homologous recombination of sister chromatids at the IS6110 sequences. Analysis of isolates representing the major M. tuberculosis lineages has revealed that the 350-kb duplication is restricted to the most recently evolved sublineages of the W/Beijing family. Within these isolates, the duplication is partly responsible for the constitutive dosR overexpression phenotype. Although the nature of the selection event giving rise to the duplication remains unresolved, its evolution is almost certainly the result of specific selective pressure(s) encountered inside the host. A preliminary in vitro screen has failed to reveal a role of the duplication in conferring resistance to common antitubercular drugs, a trait frequently associated with W/Beijing isolates. Nevertheless, this first description of a genetic remodeling event of this nature for M. tuberculosis further highlights the potential for the evolution of diversity in this important global pathogen.

Strains of the East Asian (W/Beijing) lineage of Mycobacterium tuberculosis are DosS/DosT-DosR two-component regulatory system natural mutants.

As part of our ongoing efforts to uncover the phenotypic consequences of genetic variability among clinical Mycobacterium tuberculosis isolates, we previously reported that isolates of the "East Asian" or "W/Beijing" lineage constitutively overexpress the coordinately regulated transcriptional program known as the DosR regulon under standard in vitro conditions. This phenotype distinguishes the W/Beijing lineage from all other M. tuberculosis lineages, which normally induce expression of this regulon only once exposed to low oxygen or nitric oxide, both of which result in inhibition of bacterial respiration and replication. Transcription of the DosR regulon is controlled through a two-component regulatory system comprising the transcription factor DosR and two possible cognate histidine sensor kinases, DosS and DosT. Through sequence analysis of a carefully selected set of isolates representing each of the major M. tuberculosis lineages, we describe herein a naturally occurring frameshift mutation in the gene encoding the DosT sensor kinase for isolates of the most recently evolved W/Beijing sublineages. Intriguingly, the occurrence of the frameshift mutation correlates precisely with the appearance of the constitutive DosR regulon phenotype displayed by the same "modern" W/Beijing strains. However, complementation studies have revealed that the mutation in dosT alone is not directly responsible for the constitutive DosR regulon phenotype. Our data serve to highlight the evolutionary pressure that exists among distinct M. tuberculosis lineages to maintain tight control over DosR regulon expression.

Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies.

Isolated in vitro more than half a century ago, the H37Rv strain of Mycobacterium tuberculosis still remains the strain of choice for the majority of laboratories conducting in vivo studies of TB pathogenesis. In this report we reveal that H37Rv is highly prone to losing the ability to synthesize the cell wall lipid phthiocerol dimycocerosate (PDIM) during extended periods of in vitro culture. In addition, H37Rv stocks that have been held in vitro for even a short length of time should be thought of as a heterogeneous population of PDIM-positive and PDIM-negative cell types. We demonstrate that after weekly subculture of PDIM-positive isolates over a period of 20 weeks, the proportion of PDIM-negative cells rises above 30 %. That PDIM biosynthesis is negatively selected in vitro is evident from the broad range of mutation types we observe within cultures originating from a single PDIM-positive parental clone. Moreover, the appearance of these multiple mutation types coupled with an enhanced growth rate of PDIM-negative bacteria ensures that 'PDIM-less' clones rapidly dominate in vitro cultures. It has been known for almost a decade that strains of M. tuberculosis that lack PDIM are severely attenuated during in vivo infection. Therefore, the loss of PDIM raises a very serious issue in regard to the interpretation of putative virulence factors where heterogeneous parental cultures are potentially being compared in vivo to recombinant clones isolated within a PDIM-negative background. It is essential that researchers undertaking in vivo virulence studies confirm the presence of PDIM within all recombinant clones and the parental strains they are derived from.

Major Mycobacterium tuberculosis lineages associate with patient country of origin.

Over recent years, there has been an increasing acknowledgment of the diversity that exists among Mycobacterium tuberculosis clinical isolates. To facilitate comparative studies aimed at deciphering the relevance of this diversity to human disease, an unambiguous and easily interpretable method of strain classification is required. Presently, the most effective means of assigning isolates into a series of unambiguous lineages is the method of Gagneux et al. (S. Gagneux et al., Proc. Natl. Acad. Sci. USA 103:2869-2873, 2006) that involves the PCR-based detection of large sequence polymorphisms (LSPs). In this manner, isolates are classified into six major lineages, the majority of which display a high degree of geographic restriction. Here we describe an independent replicate of the Gagneux study carried out on 798 isolates collected over a 6-year period from mostly foreign-born patients resident on the island of Montreal, Canada. The original trends in terms of bacterial genotype and patient ethnicity are remarkably conserved within this Montreal cohort, even though the patient distributions between the two populations are quite distinct. In parallel with the LSP analysis, we also demonstrate that "clustered" tuberculosis (TB) cases defined through restriction fragment length polymorphism (RFLP) analysis (for isolates with >or=6 IS6110 copies) or RFLP in combination with spoligotyping (for isolates with <6 IS6110 copies) do not stray across the LSP-defined lineage boundaries. However, our data also demonstrate the poor discriminatory power of either RFLP or spoligotyping alone for these low-IS6110-copy-number isolates. We believe that this independent validation of the LSP method should encourage researchers to adopt this system in investigations aimed at elucidating the role of strain variation in TB.

BacA, an ABC transporter involved in maintenance of chronic murine infections with Mycobacterium tuberculosis.

BacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of the bacA gene in Mycobacterium tuberculosis (Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency in M. tuberculosis did not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA in M. tuberculosis led to increased bleomycin resistance, and heterologous expression of the M. tuberculosis BacA homolog in Escherichia coli conferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.