Detection of soluble co-factor dependent protein expression in vivo : Application to the 4′-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis

ABSTRACT The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4′-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Å resolution (R = 0.229; Rfree = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM α/β structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4′-phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function.

Download Full-text

The SecA2 Secretion Factor of Mycobacterium tuberculosis Promotes Growth in Macrophages and Inhibits the Host Immune Response

Infection and Immunity ◽

10.1128/iai.01022-06 ◽

2006 ◽

Vol 74 (12) ◽

pp. 6855-6864 ◽

Cited By ~ 70

Author(s):

Sherry Kurtz ◽

Karen P. McKinnon ◽

Marschall S. Runge ◽

Jenny P.-Y. Ting ◽

Miriam Braunstein

Keyword(s):

Immune Response ◽

Mycobacterium Tuberculosis ◽

Host Immune Response ◽

Central Component ◽

Reactive Oxygen Intermediate ◽

Intracellular Growth ◽

Necrosis Factor Alpha ◽

Dependent Protein ◽

Aerosol Infection

ABSTRACT The SecA protein is present in all bacteria, and it is a central component of the general Sec-dependent protein export pathway. An unusual property of Mycobacterium tuberculosis is the presence of two SecA proteins: SecA1, the essential “housekeeping” SecA, and SecA2, the accessory secretion factor. Here, we report that a ΔsecA2 mutant of M. tuberculosis was defective for growth in the early stages of low-dose aerosol infection of C57BL/6 mice, a time during which the bacillus is primarily replicating in macrophages. Consistent with this in vivo phenotype, we found that the ΔsecA2 mutant was defective for growth in macrophages from C57BL/6 mice. The ΔsecA2 mutant was also attenuated for growth in macrophages from phox−/− mice and from NOS2−/− mice. These mice are defective in the reactive oxygen intermediate (ROI)-generating phagocyte oxidase and the reactive nitrogen intermediate (RNI)-generating inducible nitric oxide synthase, respectively. This indicated a role for SecA2 in the intracellular growth of M. tuberculosis that is independent of protecting against these ROIs or RNIs. Macrophages infected with the ΔsecA2 mutant produced higher levels of tumor necrosis factor alpha, interleukin-6, RNI, and gamma interferon-induced major histocompatibility complex class II. This demonstrated a function for M. tuberculosis SecA2 in suppressing macrophage immune responses, which could explain the role of SecA2 in intracellular growth. Our results provide another example of a relationship between M. tuberculosis virulence and inhibition of the host immune response.

Download Full-text

4′-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo

PLoS Pathogens ◽

10.1371/journal.ppat.1003097 ◽

2012 ◽

Vol 8 (12) ◽

pp. e1003097 ◽

Cited By ~ 42

Author(s):

Cécile Leblanc ◽

Thomas Prudhomme ◽

Guillaume Tabouret ◽

Aurélie Ray ◽

Sophie Burbaud ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Target ◽

Phosphopantetheinyl Transferase ◽

New Drug

Download Full-text

Induction of A- and D-type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.268.4.l625 ◽

1995 ◽

Vol 268 (4) ◽

pp. L625-L635 ◽

Cited By ~ 21

Author(s):

K. C. Bui ◽

S. Buckley ◽

F. Wu ◽

B. Uhal ◽

I. Joshi ◽

...

Keyword(s):

Protein Kinase ◽

Lung Injury ◽

Protein Expression ◽

Kinase Activity ◽

Protein Kinase Activity ◽

Short Term ◽

Dependent Protein Kinase ◽

Hyperoxic Lung Injury ◽

Dependent Protein

Hyperoxia causes a reproducible pattern of lung injury and repair in rodents, in which proliferation of alveolar epithelial cells (AEC) and fibroblasts is observed during recovery. We postulated that if quiescent cells are stimulated to reenter the cell cycle, then cyclin expression and cyclin-dependent protein kinase activity would be reactivated in AEC during the repair process after hyperoxic lung injury. To test this hypothesis, we exposed adult rats to short-term hyperoxia, followed by recovery for various times in room air. Cellular proliferation in vivo was confirmed by 1) flow cytometric analysis of DNA content (FACS) of freshly isolated AEC and 2) immunohistochemistry of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation into DNA on lung sections. The percentage of freshly isolated AEC in S phase and G2/M phase on FACS analysis increased twofold to a maximum of 16.5%, after 48 h in 100% oxygen and 48 h recovery in air. Cyclins A and D and p34cdc2 protein expression were also increased during the recovery period; while p33cdk2 and p34cdk4 increased only slightly. p34cdc2 histone H1 kinase activity, both in whole lung and in AEC, decreased initially after 48 h in oxygen. However, a marked increase in p34cdc2 kinase activity was observed at 48 h recovery in whole lung and returned to baseline by 72 h. In isolated and cultured AEC, p34cdc2 kinase activity was maximal at 24 h of recovery in air. We conclude that cyclins A and D and p34cdc2 protein expression and p34cdc2 kinase activity are increased in vivo during recovery from hyperoxic lung injury in both adult rat lungs and in AEC isolated from these lungs. We speculate that the induction of cyclin-dependent protein kinase activity is a key event in mediating the proliferative cellular repair response to lung injury.

Download Full-text

Tuberkulose - Screening

Therapeutische Umschau ◽

10.1024/0040-5930/a000181 ◽

2011 ◽

Vol 68 (7) ◽

pp. 381-387

Author(s):

Otto Schoch

Keyword(s):

Mycobacterium Tuberculosis ◽

Interferon Gamma ◽

Wird Eine ◽

Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

Download Full-text