Dissecting the Ca2+ dependence of DesA1 function in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tb) has a complex cell wall, composed largely of mycolic acids, that are crucial to its structural maintenance. The M. tb desaturase A1 (DesA1) is an essential Ca2+-binding protein that catalyses a key step in mycolic acid biosynthesis. To investigate the structural and functional significance of Ca2+ binding, we introduced mutations at key residues in its Ca2+-binding ßγ-crystallin motif to generate DesA1F303A, E304Q, and F303A-E304Q. Complementation of a conditional ΔdesA1 strain of Mycobacterium smegmatis, with the Ca2+ non-binders F303A or F303A-E304Q, failed to rescue its growth phenotype; these complements also exhibited enhanced cell wall permeability. Our findings highlight the criticality of Ca2+ in DesA1 function, and its implicit role in the maintenance of mycobacterial cellular integrity.

Herpesviral Latency-Common Themes.

Latency establishment is the hallmark feature of herpesviruses, a group of viruses, of which nine are known to infect humans. They have co-evolved alongside their hosts, and mastered manipulation of cellular pathways and tweaking various processes to their advantage. As a result, they are very well adapted to persistence. The members of the three subfamilies belonging to the family Herpesviridae differ with regard to cell tropism, target cells for the latent reservoir, and characteristics of the infection. The mechanisms governing the latent state also seem quite different. Our knowledge about latency is most complete for the gammaherpesviruses due to previously missing adequate latency models for the alpha and beta-herpesviruses. Nevertheless, with advances in cell biology and the availability of appropriate cell-culture and animal models, the common features of the latency in the different subfamilies began to emerge. Three criteria have been set forth to define latency and differentiate it from persistent or abortive infection: 1) persistence of the viral genome, 2) limited viral gene expression with no viral particle production, and 3) the ability to reactivate to a lytic cycle. This review discusses these criteria for each of the subfamilies and highlights the common strategies adopted by herpesviruses to establish latency.

The Mycobacterium tuberculosis desaturase DesA1 (Rv0824c) is a Ca2+ binding protein.

The hallmark feature of Mycobacterium tuberculosis (M.tb) the causative agent of human tuberculosis, is its complex lipid rich cell wall comprised primarily of mycolic acids, long chain fatty acids that play a key role in structural stability and permeability of the cell wall. In addition, they are involved in inhibiting phagosome-lysosome fusion and aid in granuloma formation during the pathogenic process. M.tb DesA1 is an essential acyl-acyl carrier protein desaturase predicted to catalyze the introduction of position specific double bonds during the biosynthesis of mycolic acids. This protein is one among three annotated desaturases (DesA1-3) in the M.tb genome but is unique in containing a ßγ-crystallin Greek key signature motif, a well-characterized fold known to mediate Ca2+ binding in both prokaryotic and eukaryotic organisms. Using Isothermal Titration Calorimetry and 45CaCl2 overlay, we demonstrate that Ca2+ binds to DesA1. Spectroscopic measurements suggested that this binding induces changes in protein conformation but does not lead to significant alterations in the secondary structure of the protein, a feature common to several ßγ-crystallins. An M. smegmatis strain over-expressing M.tb desA1 showed a Ca2+ dependent variation in surface phenotype, revealing a functional role for Ca2+in DesA1 activity. This study represents the first identification of a Ca2+ binding ßγ-crystallin in M.tb, emphasizing the implicit role of Ca2+ in the pathogenesis of M.tb.