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To help college health services in all parts of the country improve their approach to latent tuberculosis, two Listservs were provided for them to post their questions on dealing with TB infection. In this article, the authors present some of the questions posted in the Listservs and their corresponding answers. In their answers, the authors have tried to highlight areas of agreement, continuing controversy, and discrepancies between the Massachusetts recommendations and those of the Centers for Disease Control and Prevention and the American College Health Association. The authors conclude that a successful program of risk assessment, targeted tuberculin skin testing, and treatment of latent TB infection benefits both the individual student and the college community. Such an approach prevents cases of active TB and also uses college health service financial resources and personnel time efficiently.

The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins. signal transduction | phosphorylation motif | phosphoproteomics doi/ 10.1073/pnas.0913482107

The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes, and the products of two of these, PknA and PknB, are key components of a signal transduction pathway that regulates cell division and/or morphology. Previously, we have shown that one substrate of these kinases is Wag31, a homologue of the cell division protein DivIVA that is present, but not known to be phosphorylated, in other Gram-positive bacteria. Here, we investigate the localization and function of Wag31 and its phosphorylation. We demonstrate that Wag31 is localized to the cell poles. We further show that wag31 is an essential gene and that depletion of its product causes a dramatic morphological change in which one end of the cell becomes round rather than rod-shaped. This abnormal morphology appears to be caused by a defect in polar peptidoglycan synthesis. Finally, expression of M. tuberculosis wag31 in the wag31 conditional mutant of Mycobacterium smegmatis altered the growth rate in a manner that depended on the phospho-acceptor residue encoded by the allele being expressed. Taken together, these results indicate that Wag31 regulates cell shape and cell wall synthesis in M. tuberculosis through a molecular mechanism by which the activity of Wag31 can be modulated in response to environmental signals.

Mycobacterial SigE and SigH both initiate transcription from the sigB promoter, suggesting that they recognize similar sequences. Through mutational and primer extension analyses, we determined that SigE and SigH recognize nearly identical promoters, with differences at the 3' end of the -35 element distinguishing between SigE- and SigH-dependent promoters.

Mycobacterium leprae, a major human pathogen, grows poorly at 37[degrees]C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacteriai sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH.

The Mycobacterium tuberculosis genome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigated M. tuberculosis SigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in a sigM mutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and the kasA-kasB operon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in a sigM-disrupted strain and is undetectable in a sigM overexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions.

Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rvl139c-Rvl138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the -35 region and CGTgtc in the -10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE_PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis.

pknA and PknB are predominantly expressed during exponential growth, and the overexpression of these kinases slows growth and alters cell morphology. The data is presented that indicates that signal transduction mediated by these kinases is a novel mechanism for the regulation of cell shape in mycobacteria, one that may be conserved among gram-positive bacteria.

Mycobacterium tuberculosis, an obligate mammalian pathogen, adapts to its host during the course of infection via the regulation of gene expression. Of the regulators of transcription that play a role in this response, several alternative sigma factors of M. tuberculosis have been shown to control gene expression in response to stresses, and some of these are required for virulence or persistence in vivo. For this study, we examined the role of the alternative sigma factor SigD in M. tuberculosis gene expression and virulence. Using microarray analysis, we identified several genes whose expression was altered in a strain with a sigD deletion. A small number of these genes, including sigD itself, the gene encoding the autocrine growth factor RpfC, and a gene of unknown function, Rv1815, appear to be directly regulated by this sigma factor. By identifying the in vivo promoters of these genes, we have determined a consensus promoter sequence that is putatively recognized by SigD. The expression of several genes encoding PE-PGRS proteins, part of a large family of related genes of unknown function, was significantly increased in the sigD mutant. We found that the expression of sigD is stable throughout log phase and stationary phase but that it declines rapidly with oxygen depletion. In a mouse infection model, the sigD mutant strain was attenuated, with differences in survival and the inflammatory response in the lung between mice infected with the mutant and those infected with the wild type.

Research has been conducted on the regulation by Mycobacterium tuberculosis of the gene expression in overcoming stresses during infection. The role of sigH in the M. tuberculosis response to heat and oxidative stress has been examined and the results indicate that sigH mutant is more susceptible to the oxidative stress.

Extracytoplasmic function (ECF) sigma factors are a heterologous group of alternative sigma factors that control gene expression in response to various conditions including stress. A gene encoding the sigma factor, sigH, was cloned from Mycobacterium tuberculosis and M. smegmatis. A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including M. fortuitum and M. avium. While the M. tuberculosis and M. smegmatis sigH genes encode highly similar proteins, there are several differences in adjacent genes.

Genes encoding extracellular function (ECF) sigma factor proteins in mycobacterium were cloned and analyzed to determine their role in microbial adaptations to their extracellular environment. Southern blot analysis indicated the presence of sigE gene in Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium smegmatis. Sequence analysis of the ECF sigma factor gene indicated the presence of a highly conserved amino acid sequence that produces a protein that confers resistance to high-temperature heat shock.

Oral didanosine treatment appears to improve CD4 cell counts over the long term in some children with HIV infection. Didanosine is also known as DDI. CD4 cells are component of the immune system and are measured to evaluate immune system functioning. A group of 103 children with HIV infection, ranging in age from 6 months to 18 years, underwent didanosine therapy. Children had symptomatic HIV infection or a CD4 count of less than 500. Median length of therapy was 23 months. The CD4 count increased in 32% of patients after six months of therapy. Forty-one percent of these children improved CD4 counts for three years. The probability of survival for the 51 children with CD4 counts greater than 100 at the beginning of the study was 80% versus 39% for children with CD4 counts below that level. Treatment was discontinued in 11 children because of side effects and in 34 because of disease progression despite treatment., Background. Didanosine has demonstrated promising antiviral activity and a tolerable toxicity profile in short term studies. We describe a cohort of HIV-infected children who were treated for a prolonged period of time with didanosine. Methods. Children (6 months to 18 years of age) with symptomatic HIV infection or an absolute CD4 count < 0.5 x [10.sup.9] cells/L, received oral didanosine at doses between 20 mg/[m.sup.2] to 180 mg/[m.sup.2] every 8 hours. Clinical, immunological, and virological parameters were assessed at least every 2 months. The pharmacokinetics of didanosine were evaluated in 85 patients. Results. Previously untreated children (n = 51) and children who had received prior antiretroviral therapy (n = 52) were enrolled in the study (median time on study 22.6 months, range 2 to 48). The long-term administration of didanosine was well tolerated and no new toxicities were observed. The absolute CD4 count increased by [greater than or equal to] .05 x [10.sup.9] cells/L in 28 of 87 (32%) of patients after 6 months of therapy. Responses were also sustained in 41% of these children after 3 years of therapy. Children entering the study with a CD4 count > 0.1 x [10.sup.9] cells/L (n = 51) had a marked survival advantage (P = .00002) with an estimated survival probability after 3 years of 80% compared to 39% for children with lower CD4 counts. Although the area under the curve of didanosine increased proportionally with the dose, there was considerable interpatient variability at each dose level. There was no apparent relationship between surrogate markers of clinical outcome and plasma drug concentration. Conclusions. Didanosine was well tolerated with chronic administration, and toxicities were uncommon and usually reversible. In 41% of patients, the CD4 count increased and was maintained at the higher level even after years of treatment. Pediatrics 1994;94:724-731; HIV, didanosine, survival, toxicities, CD4 counts, pharmacokinetics, child.

Combined therapy with zidovudine (AZT) and didanosine (ddI) may benefit children with HIV infections more than either drug alone. Researchers administered varied doses of combined therapy and evaluated the outcome of 49 children who were previously untreated and 12 who had prior AZT-related toxicity. The patients tolerated AZT and ddI well at all dose levels and showed no evidence of new or enhanced toxicity. Fifty-one children gained weight while on the combined therapy program. After 24 weeks, the midpoint CD4 cell count increased from 331 to 556 cells per millimeter cubed for all patients. Among the previously untreated group, cell counts increased from 386 to 726 cells per millimeter cubed. CD4 cells are immune cells that are depleted as HIV infection progresses to advanced immune deficiency disease. Of 36 samples tested for HIV, only eight were positive after combination treatment, compared to 24 at the beginning of the study., Objective. Zidovudine and didanosine are both beneficial for the treatment of human immunodeficiency virus (HIV) infection in children. Because disease progression and toxicity often limit their long-term use as single agents, new approaches to using nucleoside analogues are necessary to improve current antiretroviral therapy. Design. We conducted a phase I-Il study to evaluate the tolerance, pharmacokinetics, and antiviral activity of the combination of zidovudine and didanosine in children with HIV infection. Sixty-eight children who were either previously untreated or who had manifested hematologic toxicity on full-dose zidovudine were enrolled. Eight dose combinations were studied in the previously untreated children, with doses of zidovudine ranging from 90 to 180 mg/[m.sup.2] every 6 hours and doses of didanosine ranging from 90 to 180 mg/[m.sup.2] every 12 hours. Results. Fifty-four previously untreated HIV-infected children were enrolled in this part of the study, of whom 49 remained in the study for a minimum of 24 weeks. For children with previous zidovudine-related hematologic toxicity, three dose levels with zidovudine at 60 mg/[m.sup.2] every 6 hours orally and didanosine ranging from 90 to 180 mg/[m.sup.2] every 12 hours orally were used. A total of 14 children were enrolled in this part of the study, and 12 remained on therapy for at least 24 weeks. No evidence of new or enhanced toxicity was observed in either group. After 24 weeks, the median CD4 cell count for all patients increased from 331 to 556 cells/[mm.sup.3] (P = .01). For the previously untreated group, the median increase in CD4 counts was from 386 to 726 cells/[mm.sup.3] (P = .003). The median p24 antigen concentration (in those with a detectable level at baseline) decreased from 95 to

A genetic analysis of microbes reveals that mycobacterial genes such as the BCG gene significantly influence genetic engineering and disease pathogenesis of mycobacterial vaccines. The Myobacterium bovis BCG gene is closely linked to the Myxococcus xanthus orotidine-5'-monophosphate decarboxylase. Vector DNA in the genome is present in all kanamycin-resistant BCG transformants.

High doses of dideoxyinosine (ddI) may cause pancreatitis in children with HIV infection. ddI is a drug used to treat HIV infection in patients who are resistant to zidovudine (AZT). Among 95 HIV-positive children between three months and 18 years old who had been treated with ddI for an average of 56 weeks, 7% developed pancreatitis. Their symptoms resolved when they stopped taking the drug. All the episodes of pancreatitis occurred in patients treated with 360 milligrams or more of ddI per square meter per day. The risk of pancreatitis recurrence is high in patients who resume treatment with a high dose of ddI. Treatment with a reduced dose lowers this risk significantly., To define predictive or contributory risk factors for pancreatitis in human immunodeficiency virus-infected children receiving dideoxyinosine (ddI), the authors evaluated 95 children, 3 months to 18 years of age, who had received ddI at 66 to 540 mg/[m.sup.2] per day for a mean of 56 weeks. Pancreatitis developed in 7 patients (7%) but resolved in all upon withdrawal of ddI. Neither age, sex, nor CD4 count at study entry was predictive of pancreatitis, but pancreatitis appeared more likely to develop in hemophiliacs than in other patients (4 of 23 vs 3 of 72). Pancreatitis developed only in patients who received ddI at the highest dose levels (7 of 60 patients who received ddI at a dose [is greater than or equal to] 360 mg/[m.sup.2] per day vs 0 of 35 patients who received [is less than or equal to]270 mg/[m.sup.2] per day). Patients in whom pancreatitis developed had received a higher mean daily dose of ddI than patients with normal amylase and lipase levels throughout the study (348 mg/[m.sup.2] vs 282 mg/[m.sup.2]), but no relationship with the cumulative dose or the duration of ddI therapy was observed. Although a statistically significant relationship between ddI plasma concentration (area under the curve) and pancreatitis was not conclusively demonstrated, as the number of patients in whom pancreatitis actually developed was small, such a relationship may have been obscured. The mean interval from initiation of ddI to onset of symptoms of pancreatitis (25 weeks) was significantly shorter than the overage duration of ddI therapy (56 weeks), and, consequently, patients in whom pancreatitis developed had been exposed to a lower cumulative dose of ddI than those with normal amylase and lipase levels (64.5 g/[m.sup.2] vs 119.06 g/[m.sup.2]). Although baseline amylase values did not correlate with the development of pancreatitis, elevation of baseline aspartate aminotransferase and alanine aminotransferase levels was significantly associated with the later development of pancreatitis. Pediatrics 1993,91:747-751, pancreatitis, dideoxyinosine, human immunodeficiency virus, antiretroviral therapy.