Interactions between Global Health Initiatives and Country Health Systems: The Case of a Neglected Tropical Diseases Control Program in Mali

Prevention of neglected tropical diseases was recently significantly scaled up in sub-Saharan Africa, protecting entire populations with mass distribution of drugs: five different diseases are now addressed simultaneously with a package of four drugs. Some argue however that, similarly to other major control programs dealing with specific diseases, this NTD campaign fails to strengthen health systems and might even negatively affect regular care provision. In 2007, we conducted an exploratory field study in Mali, observing how the program was implemented in two rural areas and how it affected the health system. At the local level, we found that the campaign effects of care delivery differed across health services. In robust and well staffed health centres, the personnel successfully facilitated mass drug distribution while running routine consultations, and overall service functioning benefitted from programme resources. In more fragile health centres however, additional program workload severely disturbed access to regular care, and we observed operational problems affecting the quality of mass drug distribution. Strong health services appeared to be profitable to the NTD control program as well as to general care

Direct Injection of Functional Single-Domain Antibodies from E. coli into Human Cells

Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs) but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in E. coli and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs) can be engineered to be injected into human cells by E. coli bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS). The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 105–106 molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of E. coli bacteria to directly deliver intracellular sdAbs (intrabodies) into human cells for analytical and therapeutic purposes

Shotgun sequencing of Yersinia enterocolitica strain W22703 (biotype 2, serotype O:9): genomic evidence for oscillation between invertebrates and mammals

<p>Abstract</p> <p>Background</p> <p><it>Yersinia enterocolitica </it>strains responsible for mild gastroenteritis in humans are very diverse with respect to their metabolic and virulence properties. Strain W22703 (biotype 2, serotype O:9) was recently identified to possess nematocidal and insecticidal activity. To better understand the relationship between pathogenicity towards insects and humans, we compared the W22703 genome with that of the highly pathogenic strain 8081 (biotype1B; serotype O:8), the only <it>Y. enterocolitica </it>strain sequenced so far.</p> <p>Results</p> <p>We used whole-genome shotgun data to assemble, annotate and analyse the sequence of strain W22703. Numerous factors assumed to contribute to enteric survival and pathogenesis, among them osmoregulated periplasmic glucan, hydrogenases, cobalamin-dependent pathways, iron uptake systems and the <it>Yersinia </it>genome island 1 (YGI-1) involved in tight adherence were identified to be common to the 8081 and W22703 genomes. However, sets of ~550 genes revealed to be specific for each of them in comparison to the other strain. The plasticity zone (PZ) of 142 kb in the W22703 genome carries an ancient flagellar cluster Flg-2 of ~40 kb, but it lacks the pathogenicity island YAPI_Ye, the secretion system <it>ysa </it>and <it>yts1</it>, and other virulence determinants of the 8081 PZ. Its composition underlines the prominent variability of this genome region and demonstrates its contribution to the higher pathogenicity of biotype 1B strains with respect to W22703. A novel type three secretion system of mosaic structure was found in the genome of W22703 that is absent in the sequenced strains of the human pathogenic <it>Yersinia </it>species, but conserved in the genomes of the apathogenic species. We identified several regions of differences in W22703 that mainly code for transporters, regulators, metabolic pathways, and defence factors.</p> <p>Conclusion</p> <p>The W22703 sequence analysis revealed a genome composition distinct from other pathogenic <it>Yersinia enterocolitica </it>strains, thus contributing novel data to the <it>Y. enterocolitica </it>pan-genome. This study also sheds further light on the strategies of this pathogen to cope with its environments.</p

Analysis of the Expression, Secretion and Translocation of the Salmonella enterica Type III Secretion System Effector SteA

Many Gram-negative pathogens possess virulence-related type III secretion systems. Salmonella enterica uses two of these systems, encoded on the pathogenicity islands SPI-1 and SPI-2, respectively, to translocate more than 30 effector proteins into eukaryotic host cells. SteA is one of the few effectors that can be translocated by both systems. We investigated the conditions affecting the synthesis of this effector, its secretion to culture media and its translocation into host cells. Whereas steA was expressed under a wide range of conditions, some factors, including low and high osmolarity, and presence of butyrate, decreased expression. SteA was efficiently secreted to the culture media under both SPI-1 and SPI-2 inducing conditions. The kinetics of translocation into murine macrophages and human epithelial cells was studied using fusions with the 3xFLAG tag, and fusions with CyaA from Bordetella pertussis. Translocation into macrophages under non-invasive conditions was mainly dependent on the SPI-2-encoded type III secretion system but some participation of the SPI-1 system was also detected 6 hours post-infection. Interestingly, both type III secretion systems had a relevant role in the translocation of SteA into epithelial cells. Finally, a deletion approach allowed the identification of the N-terminal signal necessary for translocation of this effector. The amino acid residues 1–10 were sufficient to direct translocation into host cells through both type III secretion systems. Our results provide new examples of functional overlapping between the two type III secretion systems of Salmonella

Detection and Functional Characterization of a 215 Amino Acid N-Terminal Extension in the Xanthomonas Type III Effector XopD

During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD1-760) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD216-760). Furthermore, the N-terminal extension of XopD, which is absent in XopD216-760, is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta

Functional and Computational Analysis of Amino Acid Patterns Predictive of Type III Secretion System Substrates in Pseudomonas syringae

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant pathogen Pseudomonas syringae pathovar tomato strain DC3000 possess characteristic patterns, including (i) greater than 10% serine within the first 50 amino acids, (ii) an aliphatic residue or proline at position 3 or 4, and (iii) a lack of acidic amino acids within the first 12 residues. Here, the functional significance of the P. syringae T3SS substrate compositional patterns was tested. A mutant AvrPto effector protein lacking all three patterns was secreted into culture and translocated into plant cells, suggesting that the compositional characteristics are not absolutely required for T3SS targeting and that other recognition mechanisms exist. To further analyze the unique properties of T3SS targeting signals, we developed a computational algorithm called TEREE (Type III Effector Relative Entropy Evaluation) that distinguishes DC3000 T3SS substrates from other proteins with a high sensitivity and specificity. Although TEREE did not efficiently identify T3SS substrates in Salmonella enterica, it was effective in another P. syringae strain and Ralstonia solanacearum. Thus, the TEREE algorithm may be a useful tool for identifying new effector genes in plant pathogens. The nature of T3SS targeting signals was additionally investigated by analyzing the N-terminus of FtsX, a putative membrane protein that was classified as a T3SS substrate by TEREE. Although the first 50 amino acids of FtsX were unable to target a reporter protein to the T3SS, an AvrPto protein substituted with the first 12 amino acids of FtsX was translocated into plant cells. These results show that the T3SS targeting signals are highly mutable and that secretion may be directed by multiple features of substrates

Modified Needle-Tip PcrV Proteins Reveal Distinct Phenotypes Relevant to the Control of Type III Secretion and Intoxication by Pseudomonas aeruginosa

The type III secretion system (T3SS) is employed to deliver effector proteins to the cytosol of eukaryotic hosts by multiple species of Gram-negative bacteria, including Pseudomonas aeruginosa. Translocation of effectors is dependent on the proteins encoded by the pcrGVHpopBD operon. These proteins form a T3S translocator complex, composed of a needle-tip complex (PcrV), translocons (PopB and PopD), and chaperones (PcrG and PcrH). PcrV mediates the folding and insertion of PopB/PopD in host plasmic membranes, where assembled translocons form a translocation channel. Assembly of this complex and delivery of effectors through this machinery is tightly controlled by PcrV, yet the multifunctional aspects of this molecule have not been defined. In addition, PcrV is a protective antigen for P. aeruginosa infection as is the ortholog, LcrV, for Yersinia. We constructed PcrV derivatives containing in-frame linker insertions and site-specific mutations. The expression of these derivatives was regulated by a T3S-specific promoter in a pcrV-null mutant of PA103. Nine derivatives disrupted the regulation of effector secretion and constitutively released an effector protein into growth medium. Three of these regulatory mutants, in which the linker was inserted in the N-terminal globular domain, were competent for the translocation of a cytotoxin, ExoU, into eukaryotic host cells. We also isolated strains expressing a delayed-toxicity phenotype, which secrete translocators slowly despite the normal level of effector secretion. Most of the cytotoxic translocation-competent strains retained the protective epitope of PcrV derivatives, and Mab166 was able to protect erythrocytes during infection with these strains. The use of defined PcrV derivatives possessing distinct phenotypes may lead to a better understanding of the functional aspects of T3 needle-tip proteins and the development of therapeutic agents or vaccines targeting T3SS-mediated intoxication

Accurate Prediction of Secreted Substrates and Identification of a Conserved Putative Secretion Signal for Type III Secretion Systems

The type III secretion system is an essential component for virulence in many Gram-negative bacteria. Though components of the secretion system apparatus are conserved, its substrates—effector proteins—are not. We have used a novel computational approach to confidently identify new secreted effectors by integrating protein sequence-based features, including evolutionary measures such as the pattern of homologs in a range of other organisms, G+C content, amino acid composition, and the N-terminal 30 residues of the protein sequence. The method was trained on known effectors from the plant pathogen Pseudomonas syringae and validated on a set of effectors from the animal pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) after eliminating effectors with detectable sequence similarity. We show that this approach can predict known secreted effectors with high specificity and sensitivity. Furthermore, by considering a large set of effectors from multiple organisms, we computationally identify a common putative secretion signal in the N-terminal 20 residues of secreted effectors. This signal can be used to discriminate 46 out of 68 total known effectors from both organisms, suggesting that it is a real, shared signal applicable to many type III secreted effectors. We use the method to make novel predictions of secreted effectors in S. Typhimurium, some of which have been experimentally validated. We also apply the method to predict secreted effectors in the genetically intractable human pathogen Chlamydia trachomatis, identifying the majority of known secreted proteins in addition to providing a number of novel predictions. This approach provides a new way to identify secreted effectors in a broad range of pathogenic bacteria for further experimental characterization and provides insight into the nature of the type III secretion signal