Identification of Mycobacterium tuberculosis clinical isolates with altered phagocytosis by human macrophages due to a truncated lipoarabinomannan

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.     

A cluster of basic amino acid residues in the gamma370-381 sequence of fibrinogen comprises a binding site for platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa)

Adhesive interactions of platelet integrin alpha(IIb)beta3 with fibrinogen and fibrin are central events in hemostasis and thrombosis. However, the mechanisms by which alpha(IIb)beta3 binds these ligands remain incompletely understood. We have recently demonstrated that alpha(IIb)beta3 binds the gamma365-383 sequence in the gammaC-domain of fibrin(ogen). This sequence contains neither the AGDV nor the RGD recognition motifs, known to bind alpha(IIb)beta3, suggesting the different specificity of the integrin. Here, using peptide arrays, mutant fibrinogens, and recombinant mutant gammaC-domains, we have examined the mechanism whereby alpha(IIb)beta3 binds gamma365-383. The alpha(IIb)beta3-binding activity was localized within gamma370-381, with two short sequences, gamma370ATWKTR375 and gamma376WYSMKK381, being able to independently bind the integrin. Furthermore, recognition of alpha(IIb)beta3 by gamma370-381 depended on four basic residues, Lys373, Arg375, Lys380, and Lys381. Simultaneous replacement of these amino acids and deletion of the gamma408AGDV411 sequence in the recombinant gammaC-domain resulted in the loss of alpha(IIb)beta3-mediated platelet adhesion. Confirming the critical roles of the identified residues, abnormal fibrinogen Kaiserslautern, in which gammaLys380 is replaced by Asn, demonstrated delayed clot retraction and impaired alpha(IIb)beta3 binding. Also, a mutant recombinant fibrinogen modeled after the naturally occurring variant Osaka V (gammaArg375 --> Gly) showed delayed clot retraction and reduced binding to purified alpha(IIb)beta3. These results identify the gamma370-381 sequence of fibrin(ogen) as the binding site for alpha(IIb)beta3 involved in platelet adhesion and clot retraction and define the new recognition specificity of this integrin.     

An additional GerE-controlled gene encoding an abundant spore coat protein from Bacillus subtilis.

We describe the identification and characterization of a gene, herein designated cotG, encoding an abundant coat protein from the spores of Bacillus subtilis. The cotG open reading frame is 195 codons in length and is capable of encoding a polypeptide of 24 kDa that contains nine tandem copies of the 13-amino-acid long, approximately repeated sequence H/Y-K-K-S-Y-R/C-S/T-H/Y-K-K-S-R-S. cotG is located at 300 degrees on the genetic map close to another coat protein gene, cotB. The cotG and cotB genes are in divergent orientation and are separated by 1.3 kb. Like the promoter for cotB, the cotG promoter is induced at a late stage of sporulation under the control of the RNA polymerase sigma factor sigma K and the DNA-binding protein GerE. The -10 and -35 nucleotide sequences of the cotG promoter resemble those of other promoters recognized by sigma K-containing RNA polymerase, and centered 70 bp upstream of the apparent start site is a sequence that matches the consensus binding site for GerE. Spore coat proteins from a newly constructed cotG null mutant lack not only CotG but also CotB, a finding that suggests that CotG may be a morphogenetic protein that is required for the incorporation of CotB into the coat.     

Assembly and function of a spore coat-associated transglutaminase of Bacillus subtilis

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains epsilon-(gamma-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under sigma(K) control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.     

Phylogeographic Pattern and Extensive Mitochondrial DNA Divergence Disclose a Species Complex within the Chagas Disease Vector Triatoma dimidiata

Background

Triatoma dimidiata is among the main vectors of Chagas disease in Latin America. However, and despite important advances, there is no consensus about the taxonomic status of phenotypically divergent T. dimidiata populations, which in most recent papers are regarded as subspecies.

Methodology and Findings

A total of 126 cyt b sequences (621 bp long) were produced for specimens from across the species range. Forty-seven selected specimens representing the main cyt b clades observed (after a preliminary phylogenetic analysis) were also sequenced for an ND4 fragment (554 bp long) and concatenated with their respective cyt b sequences to produce a combined data set totalling 1175 bp/individual. Bayesian and Maximum-Likelihood phylogenetic analyses of both data sets (cyt b, and cyt b+ND4) disclosed four strongly divergent (all pairwise Kimura 2-parameter distances >0.08), monophyletic groups: Group I occurs from Southern Mexico through Central America into Colombia, with Ecuadorian specimens resembling Nicaraguan material; Group II includes samples from Western-Southwestern Mexico; Group III comprises specimens from the Yucatán peninsula; and Group IV consists of sylvatic samples from Belize. The closely-related, yet formally recognized species T. hegneri from the island of Cozumel falls within the divergence range of the T. dimidiata populations studied.

Conclusions

We propose that Groups I–IV, as well as T. hegneri, should be regarded as separate species. In the Petén of Guatemala, representatives of Groups I, II, and III occur in sympatry; the absence of haplotypes with intermediate genetic distances, as shown by multimodal mismatch distribution plots, clearly indicates that reproductive barriers actively promote within-group cohesion. Some sylvatic specimens from Belize belong to a different species – likely the basal lineage of the T. dimidiata complex, originated ∼8.25 Mya. The evidence presented here strongly supports the proposition that T. dimidiata is a complex of five cryptic species (Groups I–IV plus T. hegneri) that play different roles as vectors of Chagas disease in the region.     

Detection of Oseltamivir-Resistant Pandemic Influenza A(H1N1)pdm2009 in Brazil: Can Community Transmission Be Ruled Out?

Although surveillance efforts that monitor the emergence of drug-resistant strains of influenza are critical, systematic analysis is overlooked in most developing countries. We report on the occurrence of strains of pandemic influenza A(H1N1)pdm09 with resistance and decreased susceptibility to oseltamivir (OST) in Brazil in 2009, 2011 and 2012. We found 7 mutant viruses, 2 with the mutation S247N and other 5 with the mutation H275Y. Most of these viruses were from samples concentrated in the southern region of Brazil. Some of these resistant viruses were detected prior to the initiation of OST treatment, suggesting that community transmission of mutant viruses may exist. Moreover, we show that one of these OST-resistant (H275Y) strains of A(H1N1)pdm09 was discovered in the tri-border region between Brazil, Argentina and Paraguay, highlighting that this strain could also be found in other Latin American countries. Our findings reinforce the importance of enhanced antiviral resistance surveillance in Brazil and in other Latin American countries to confirm or rule out the community transmission of OST-resistant strains of A(H1N1)pdm09.     

Sensitivity to stress in the bivalve Macoma balthica from the most northern (Arctic) to the most southern (French) populations: low sensitivity in Arctic populations because of genetic adaptations?

The stress sensitivity, determined in copper exposureexperiments and in survival in air tests, and thegenetic structure, measured by means of isoenzymeelectrophoresis, were assessed in populations of theBaltic clam Macoma balthica (L.) from itssouthern to its northern distribution limit, in orderto test the hypotheses that near the distributionlimit the clams would be more stress sensitive andwould have a lower genetic variability. Thepopulations in west and north Europe show a stronggenetic resemblance. The populations in the sub-ArcticWhite Sea are genetically slightly different, and showa low stress sensitivity. The populations in theArctic Pechora Sea are genetically very distant fromthe other populations, and show the lowest stresssensitivity. Near the southern distribution limit, inagreement with the hypotheses, genetic variability islow and stress sensitivity high. On the other hand, incontrast to expectation, near the northerndistribution limit, in the populations of the PechoraSea, the genetic variability was higher, thus notreduced, and the stress sensitivity was low comparedto all other populations. Yet, it remains a questionif such is due to gradual physiologicalacclimatization (and ongoing differential selection)or to genetic adaptation.