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SummaryIn previously published work, we identified three Mycobacterium tuberculosis sigma (s) factor genes responding to heat shock (sigB, sigE and sigH ). Two of them (sigB and sigE ) also responded to SDS exposure. As these responses to stress suggested that the s factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence. In this work, we characterize a sigE mutant of M. tuberculosis H37Rv. The sigE mutant strain was more sensitive than the wild-type strain to heat shock, SDS and various oxidative stresses. It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild-type strain to the killing activity of activated murine macrophages. Using microarray technology and quantitative reverse transcription -polymerase chain reaction (RT -PCR), we started to define the s E regulon of M. tuberculosis and its involvement in the global regulation of the stress induced by SDS. We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock. We also identified several genes that are no longer induced when s E is absent. These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins.

On the 21st of February 2020 a resident of the municipality of Vo, a small town near Padua, died of pneumonia due to SARS-CoV-2 infection. This was the first COVID-19 death detected in Italy since the emergence of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days. We collected information on the demography, clinical presentation, hospitalization, contact network and presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo at two consecutive time points. On the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI) 2.1-3.3%). On the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI 0.8-1.8%). Notably, 43.2% (95% CI 32.2-54.7%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic. The mean serial interval was 6.9 days (95% CI 2.6-13.4). We found no statistically significant difference in the viral load (as measured by genome equivalents inferred from cycle threshold data) of symptomatic versus asymptomatic infections (p-values 0.6 and 0.2 for E and RdRp genes, respectively, Exact Wilcoxon-Mann-Whitney test). Contact tracing of the newly infected cases and transmission chain reconstruction revealed that most new infections in the second survey were infected in the community before the lockdown or from asymptomatic infections living in the same household. This study sheds new light on the frequency of asymptomatic SARS-CoV-2 infection and their infectivity (as measured by the viral load) and provides new insights into its transmission dynamics, the duration of viral load detectability and the efficacy of the implemented control measures.

SummaryThe ability of Mycobacterium tuberculosis to adapt to different environments in the infected host is essential for its pathogenicity. Consequently, this organism must be able to modulate gene expression to respond to the changing conditions it encounters during infection. In this paper we begin a comprehensive study of M. tuberculosis gene regulation, characterizing the transcript levels of 10 of its 13 putative sigma factor genes. We developed a real-time RT-PCR assay using a family of novel fluorescent probes called molecular beacons to quantitatively measure the different mRNAs. Three sigma factor genes were identified that have increased mRNA levels after heat shock, two of which also responded to detergent stress. In addition, we also identified a sigma factor gene whose mRNA increased after mild cold shock and a second that responded to conditions of low aeration.

SummaryLike other bacterial species, Mycobacterium tuberculosis has multiple sigma (s) factors encoded in its genome. In previously published work, we and others have shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and, in some cases, cause attenuated virulence phenotypes. In this paper, we characterize a M. tuberculosis mutant lacking the ECF s factor s H . This mutant was more sensitive than the wild type to heat shock and to various oxidative stresses, but did not show decreased ability to grow inside macrophages. Using quantitative reverse transcription-PCR and microarray technology, we have started to define the s H regulon and its involvement in the global regulation of the response to heat shock and the thiol-specific oxidizing agent diamide. We identified 48 genes whose expression increased after exposure of M. tuberculosis to diamide; out of these, 39 were not induced in the sigH mutant, showing their direct or indirect dependence on s H . Some of these genes encode proteins whose predicted function is related to thiol metabolism, such as thioredoxin, thioredoxin reductase and enzymes involved in cysteine and molybdopterine biosynthesis. Other genes under s H control encode transcriptional regulators such as sigB, sigE, and sigH itself.

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zur operon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated a zur mutant in M. tuberculosis, examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zurregulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.Mycobacterium tuberculosis is a human pathogen that infects and replicates within macrophages. This microorganism lives in phagosomes that fail to fuse with lysosomes and has adapted its lifestyle to survive and replicate in the changing environment within the endosomal system (20).The long-recognized phenomenon of nutritional immunity, in which sequestration of iron and possibly other metals occurs as a nonspecific host response to infection (2), hints in general terms at the possibility of a keen competition between host and parasite for essential metal ions. A critical point is the bacterial ability to compete with the host for nutrients, and the acquisition of metal ions has important implications for intracellular survival.Pathogenic bacteria respond to such limitations by inducing metabolic functions that overcome nutritional deficiencies and/or inducing virulence functions required for immediate survival and spread to subsequent anatomical sites of infection. The outcome of this competition between the host cell and the microorganism is certainly one of the most important factors determining the ability of pathogens to multiply and cause disease (41).Metalloregulatory proteins sense the intracellular levels of specific metal ions and mediate a transcriptional response aimed at restoring homeostasis when these levels are altered. In prokaryotes, these transcriptional regulators are clustered...

The nucleotide sequence of comC, the gene encoding the 17-residue competence-stimulating peptide (CSP) of Streptococcus pneumoniae (L. S. Håvarstein, G. Coomaraswamy, and D. A. Morrison, Proc. Natl. Acad. Sci. USA 92:11140-11144, 1995) was determined with 42 encapsulated strains of different serotypes. A new allele, comC2, was found in 13 strains, including the type 3 Avery strain, A66, while all others carried a gene (now termed comC1) identical to that originally described for strain Rx1. The predicted mature product of comC2 is also a heptadecapeptide but differs from that of comC1 at eight residues. Both CSP-1 and CSP-2 synthetic peptides were used to induce competence in the 42 strains; 48% of the strains became competent after the addition of the synthetic peptide, whereas none were transformable without the added peptides.Genetic transformation not only is a valuable tool for molecular genetic analysis of Streptococcus pneumoniae (pneumococcus) but also appears to play a significant role in the evolution of this species, as in the assembly of mosaic antibiotic resistance genes containing blocks of information from other bacteria (6, 17) and in mediating a rapid mixing of alleles among natural populations (4). Yet, although transformation has long been described as a characteristic of the pneumococci, as a practical matter most studies involving this process have focused on a few laboratory strains, mainly descended from a single unencapsulated subclone (R36A) (2), for which optimal media and protocols were developed. Indeed, it has never been established just what proportion of pneumococcal isolates is transformable. This fraction has been difficult to assess, both because the pneumococcal capsule reduces or abolishes competence for genetic transformation (3,5,7,14,20,25) and because the optimal conditions for competence vary between strains.Competence for transformation in pneumococcus is not constitutive but is regulated by a quorum-sensing pheromone signal (23, 24). It was previously shown that culture supernatants from one strain, Rx1 (16), activate some other strains to competence and can induce competence in some encapsulated strains but not in others, including the type 3 strain A66 (25), even though an unencapsulated derivative of that strain is transformable (22). Following the recent identification of a small peptide from strain Rx1 with competence-stimulating activity (a competence-stimulating peptide [CSP]) (12), we reinvestigated the paradoxical behavior of A66, as a pure pheromone might be more effective than the crude culture supernatants used previously, but the results were still negative. We now trace the unresponsive character of strain A66 to the competence pheromone regulatory circuit itself and show that it reflects allelic variation of the pheromone gene in different isolates of this species.Failure of A66 to respond to the CSP of Rx1. Strain Rx1 is descended from R36A, an unencapsulated transformable derivative of D39S, a type 2 clinical isolate that was employed in early studies o...

Pathogenetic processes that facilitate the entry, replication, and persistence of Mycobacterium tuberculosis (MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection. Identification of genes that are differentially expressed in vivo would provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited. In this study, the levels of selected MTB mRNAs were quantified in vitro in axenic culture, in vivo in the lungs of mice, and in lung specimens obtained from TB patients with active disease. We report the differential expression of MTB mRNAs associated with iron limitation, alternative carbon metabolism, and cellular hypoxia, conditions that are thought to exist within the granulomatous lesions of TB, in the lungs of wild-type C57BL͞6 mice as compared with bacteria grown in vitro. Analysis of the same set of mRNAs in lung specimens obtained from TB patients revealed differences in MTB gene expression in humans as compared with mice.T he clinical course of tuberculosis (TB) comprises a sequence of different stages (1). Airborne bacilli that implant in the lung alveoli are phagocytosed by alveolar macrophages (2). Intracellular replication of Mycobacterium tuberculosis (MTB) results in a primary lesion, followed by lymphohematogenous dissemination and secondary lesions in the lungs and other organs. In most individuals, disease progression is arrested at this stage by the acquired immune response, and a clinically latent state ensues. Postprimary disease arises from the subsequent reactivation of dormant tuberculous foci (3, 4). For the nearly 2 billion individuals worldwide who have been infected with MTB, the lifetime risk of developing TB is Ϸ10% (5). In the absence of effective treatment, TB case fatality rates can exceed 50%, as evidenced by longitudinal clinical studies in the preantimicrobial era (6).Despite the global importance of TB as a leading cause of morbidity and mortality, little is known about the host-pathogen interactions at each stage of infection in humans. This information has been difficult to obtain because medical ethics demand that anti-TB therapy be initiated immediately on diagnosis. The uninterrupted course of disease can be studied in TB patients who are unresponsive to therapy, typically due to multidrugresistant MTB. In such cases, palliative resection of diseased tissue is sometimes undertaken (7). Resected human tissues, which would otherwise be discarded, can be used to analyze host-pathogen interactions in situ (8, 9).Adaptation of MTB to environmental changes in the course of infection is likely mediated by differential gene expression. An important role for transcriptional regulation in the life cycle of MTB is suggested by genes encoding 13 RNA polymerase -factors and Ͼ100 putative regulatory proteins in the genome (10). Although the cellular processes controlled by these transcription f...