The Inactivation of a New Peptidoglycan Hydrolase Pmp23 Leads to Abnormal Septum Formation in Streptococcus pneumoniae

The bacterial peptidoglycan is the major component of the cell wall which integrity is essential to cell survival. In a previous work, we identified, in the positive-Gram pathogen Streptococcus pneumoniae , a unique protein containing a new putative peptidoglycan hydrolytic domain named PECACE (PEptidoglycan CArbohydrate Cleavage Enzyme). In this study, we characterise the physiological function of this protein called Pmp23 (Pneumococcal Membrane Protein of 23 kDa). A cell wall hydrolytic activity is observed with the recombinant protein. Inactivation of the pmp23 gene in the pneumococcus led to a decreased flocculation, an increased sensitivity to β-lactam antibiotics and morphological alterations affecting the formation and localisation of the division septa. Taken together these observations indicate that Pmp23 is a hydrolase whose function is linked to peptidoglycan metabolism at the septum site

Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface

The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence

Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria

Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins

A unique formalism for VLSI logical and electrical synthesis

This paper presents a unique formalism of knowledge representation of a MOS digital designer in terms of sets of rewriting rules. This formalism provides solution to logical synthesis from functional description to gate network, to electrical synthesis from gate network to equivalent MOS transistor network and to logical extraction from a transistor network. Corresponding inference origines extensively use a general commutative unification process with variable arity of operators