Imbalance in seminal fluid MIF indicates male infertility

Macrophage migration inhibitory factor (MIF) is a ubiquitous cytokine that functions in reproduction and plays an important role in sperm maturation and motility. Here we reveal a correlation between MIF levels in human seminal fluid and fertility status. We identify an abnormal biphasic profile of MIF in the seminal fluid of patients with impaired sperm parameters. Our findings may be of interest for the development of a diagnostic method for fertility status.     

A Deficiency in Arabinogalactan Biosynthesis Affects Corynebacterium glutamicum Mycolate Outer Membrane Stability

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum. AftB catalyzes the transfer of Araf (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal β(1 → 2)-linked Araf residues. Here we show that ΔaftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a ΔaftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum, but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae, and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the ΔaftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum.Corynebacterineae is a specific suborder of Gram-positive bacteria that includes medically and economically important species, such as Mycobacterium tuberculosis, Mycobacterium leprae, and Corynebacterium glutamicum. The ultrastructure of cell envelopes in the Corynebacterineae has been intensively studied during recent decades and has revealed a completely unexpected scheme. Indeed, they are composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. This outer membrane is made up of mycolic acids that either esterify trehalose (free mycolates) or are terminal Araf residues of AG skeleton (bound mycolates) (38-40). The disclosure of this very atypical structure initially came from functional studies in which pore-forming proteins were identified in almost all members of the Corynebacterineae (41, 62), pointing out the presence of an unexpected additional hydrophobic barrier in the cell envelope of these Gram-positive bacteria. Its presence was recently clearly visualized by cryo-electron microscopy of vitreous sections (CEMOVIS) (29, 67), but the exact physiological properties of this barrier are poorly documented because only a few integral outer membrane proteins (OMPs) are yet characterized. In mycobacteria, two examples of integral OMPs are well documented: OmpA of M. tuberculosis and MspA of Mycobacterium smegmatis. MspA is a major porin involved in mycolate outer membrane permeability (58, 64) and has a very specific structure based on a β-barrel core reminiscent of that found in Gram-negative porins (25). In C. glutamicum, four different porins of very low molecular mass (PorA [36, 42], PorH [30], and PorB and PorC [18]) have been identified. Very recently it was shown that a heterooligomeric structure composed of PorA and PorH is needed to form the major cell wall channel of C. glutamicum (6). Although these proteins have been extensively characterized in vitro, their physiological importance remains elusive. Surprisingly, preliminary structural studies of PorB suggested that it could be organized in an α-helical structure, in contrast to all the “canonical” porins, and thus could represent a new class of outer membrane proteins (66).Because Gram-negative outer membrane proteins and MspA from M. smegmatis are organized in β-barrel structures, two studies aimed to identify M. tuberculosis OMPs by a bioinformatic approach using β-barrel prediction algorithms (43, 57). Although very attractive, this approach has several limitations. Indeed, these algorithms generally produce a high number of false-positive proteins and are not able to predict amphipathic α-helical structures such as those expected for PorB. Nevertheless, this approach led to the identification of a new channel-forming outer membrane protein of M. tuberculosis, Rv1698, which is conserved among all members of the Corynebacterineae (55). This protein increases the susceptibility of bacteria to hydrophilic antibiotics and increases the rate of glucose uptake. Its structure has not yet been characterized.Isolation of the outer membrane of mycobacteria or corynebacteria using standard biochemical methods is difficult due to the covalent links between mycolic acids and the underlying heteropolymer hampering the identification of new OMPs. In order to overcome this problem, an alternative approach could come from outer membrane-derived vesicles (OMVs), whose release in the medium is a conserved mechanism among Gram-negative bacteria and has been observed in many environments (34). Increased OMV release has been reported for mutants lacking either components of the Tol-Pal system (8, 63) or Lpp, the major lipoprotein involved in noncovalent interactions between the outer membrane and the peptidoglycan (8, 14, 15, 22, 31). This suggested that OMV production in Gram-negative bacteria is controlled through specific domains that promote outer membrane protein-peptidoglycan and outer membrane protein-inner membrane interactions (22). OMVs from Gram-negative bacteria are composed of lipopolysaccharide (LPS), phospholipids, and outer membrane proteins but lack inner membrane proteins. In some cases, elementary units of peptidoglycan and periplasmic proteins are also recovered in these vesicles (35, 37).C. glutamicum is the prototype of amino acid producers and has been widely used in biotechnology for several decades. More recently it was also shown to be a very attractive model for depicting cell wall biosynthesis of the Corynebacterineae (45). Indeed, an extensive library of mutants involved in the different steps of mycolic acid and arabinogalactan synthesis is available for C. glutamicum but not for mycobacteria, where these components are strictly essential for viability. In this work, we wanted to screen different C. glutamicum cell wall mutants for their ability to release outer membrane-derived vesicles in the external medium. More particularly, we focused on mutants with altered covalent linkage between the mycolate outer membrane and the arabinogalactan. Accordingly, we constructed an arabinosyltransferase mutant unable to catalyze the transfer of Araf onto the arabinan domain of AG to form terminal β(1 → 2)-linked Araf residues. This abnormal AG structure results in both a decrease in mycoloylation sites on arabinogalactan and a concomitant appearance of outer membrane-derived fragments in the external medium.     

Comparative Evaluation of MRSA Nasal Colonization Epidemiology in the Urban and Rural Secondary School Community of Kurdistan,Iraq

BackgroundTo study the nasal carriage rate of Staphylococcus aureus (S. aureus) (including methicillin-resistant strains) in secondary school community of the urban and rural districts of the Kurdistan region of Iraq, a cross-sectional population based survey was carried out in the city Duhok and rural areas of Amedya, Akre and Zakho.MethodsNasal swabs were obtained from nostrils of 509 students aged 14-23 years. Resistance to methicillin was assessed by Kirby-Bauer disk diffusion and agar dilution assay. Vancomycin sensitivity was also tested on Muller-Hinton agar.ResultsIt was found that the frequency of overall S. aureus nasal carriage (SANC) was 17.75% (90/509, CI₉₅, 14.58–21.42%). In urban areas, the carriage rate was 20.59% (49/239, CI₉₅, 15.64–26.29%), whereas it was 15.24% (41/270, CI₉₅, 11.17–20.10%) in rural districts. The frequency of methicillin-resistant S. aureus (MRSA) among the isolated strains was found to be 2.04% (1/49) and 21.95% (9/41) in urban and rural areas respectively. It was found that in urban residents, the odd ratio (OR) of acquiring SANC was 1.44 (CI₉₅, 0.91-2.27%) and risk ratio (RR) was at least 1.35 (CI₉₅, 0.92-1.96%) while OR decreased to 0.12 (CI₉₅, 0.01-0.96%) for MRSA carriage. Hence, the S. aureus carriage rate was higher in urban districts compared to rural areas while more MRSA were found in rural areas compared to urban districts. All studied strains were sensitive to vancomycin.ConclusionThis study provided baseline information for S. aureus nasal colonization in the region. Also, it showed that living in rural areas increased the odds of MRSA colonization. More attention should be paid to control MRSA colonization in rural communities.     

Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85

Mycolic acids, long-chain (C70-C90) alpha-alkyl, beta-hydroxy fatty acids, are characteristic cell envelope components of mycobacteria; similar but shorter-chain substances occur in corynebacteria and related taxa. These compounds apparently play an important role in the physiology of these bacteria. The deduced N-terminal region of PS1, one of the two major secreted proteins of Corynebacterium glutamicum encoded by the csp1 gene, is similar to the antigens 85 complex of Mycobacterium tuberculosis which has been shown to be associated in vitro with a mycoloyltransferase activity onto trehalose. Overexpression of PS1 in the wild-type strain of C. glutamicum suggested the implication of the protein in the transfer of corynomycolates, evidenced by an increase esterification of the cell wall arabinogalactan with corynomycolic acid residues and an accumulation of trehalose dicorynomycolates. Overexpression of truncated forms of PS1 demonstrated that the crucial region for transfer activity of the protein involves all the region of homology with antigens 85. To establish the putative mycoloyltransferase activity of PS1, a csp1-inactivated mutant of C. glutamicum was biochemically characterized. Inactivation of the gene resulted in: (i) a 50% decrease in the cell wall corynomycolate content; (ii) the alteration of the permeability of the C. glutamicum cell envelope; (iii) the decrease of the trehalose dicorynomycolate content; (iv) the accumulation of trehalose monocorynomycolate; and (v) the appearance of a glycolipid identified as 6-corynomycoloylglucose. Complementation of the mutant by the csp1 gene fully restored the wild-type phenotype. Finally, a mycoloyltransferase assay established that PS1 possesses a trehalose mycoloyltransferase activity. To define the in vivo function of antigens 85, the csp1-inactivated mutant was complemented with the fbpA, fbpB or fbpC genes. Complementation with the different fbp genes restored the normal cell wall corynomycolate content and permeability, but did not affect either the fate of trehalose corynomycolates or the occurrence of glucose corynomycolate. Thus, PS1 is one of the enzymes that transfer corynomycoloyl residues onto both the cell wall arabinogalactan and trehalose monocorynomycolate, whereas in the whole bacterium the mycobacterial antigens 85A, 85B and 85C can transfer mycolates only onto the cell wall acceptor in C. glutamicum.     

Photoaffinity cross-linking of acyl carrier protein to Escherichia coli membranes.

Acyl Carrier Protein (ACP) is a small acidic protein which interacts with the various enzymes implicated in the biosynthesis of fatty acids in E. coli. It also interacts with the inner membrane proteins implicated in the biosynthesis of phospholipids. Samples of radioactive ACP were prepared with high specific activities and bearing photoactivable aryl azide derivatives. Two photoactivable reagents were used: para azido phenacyl bromide (pAPA) which reacts with the SH of the ACP prosthetic group and the N-hydroxysuccinimide ester of 4-azido salicylic acid (NHS-ASA) which reacts with the amino groups of the protein. Various methods were used to demonstrate that ACP could be cross-linked specifically to an inner membrane protein of E. coli, most probably to the glycerol-3-phosphate acyl transferase (GPAT). This covalent link should provide a powerful tool for further analysis of the structure of GPAT and its role in phospholipid biosynthesis. These photoactivable aryl azide derivatives of ACP could also be very useful for studying the interaction of ACP with the soluble enzymes implicated in fatty acid biosynthesis.     

Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis

The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.     

The ColV, I-K94 plasmid and heat sensitivity of Escherichia coli

The ColV, I-K94 plasmid markedly increased the heat sensitivity of Escherichia coli K12 but had no sensitizing effect on a wild E. coli isolate. The plasmid effect on strain K12 appeared to result from ColV-encoded transfer and colicin components possibly due to their effects on membrane properties.