| 1. |
- Ademuyiwa, Adesoji O., et al.
(författare)
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Determinants of morbidity and mortality following emergency abdominal surgery in children in low-income and middle-income countries
- 2016
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Ingår i: BMJ Global Health. - : BMJ Publishing Group Ltd. - 2059-7908. ; 1:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Child health is a key priority on the global health agenda, yet the provision of essential and emergency surgery in children is patchy in resource-poor regions. This study was aimed to determine the mortality risk for emergency abdominal paediatric surgery in low-income countries globally.Methods: Multicentre, international, prospective, cohort study. Self-selected surgical units performing emergency abdominal surgery submitted prespecified data for consecutive children aged <16 years during a 2-week period between July and December 2014. The United Nation's Human Development Index (HDI) was used to stratify countries. The main outcome measure was 30-day postoperative mortality, analysed by multilevel logistic regression.Results: This study included 1409 patients from 253 centres in 43 countries; 282 children were under 2 years of age. Among them, 265 (18.8%) were from low-HDI, 450 (31.9%) from middle-HDI and 694 (49.3%) from high-HDI countries. The most common operations performed were appendectomy, small bowel resection, pyloromyotomy and correction of intussusception. After adjustment for patient and hospital risk factors, child mortality at 30 days was significantly higher in low-HDI (adjusted OR 7.14 (95% CI 2.52 to 20.23), p<0.001) and middle-HDI (4.42 (1.44 to 13.56), p=0.009) countries compared with high-HDI countries, translating to 40 excess deaths per 1000 procedures performed.Conclusions: Adjusted mortality in children following emergency abdominal surgery may be as high as 7 times greater in low-HDI and middle-HDI countries compared with high-HDI countries. Effective provision of emergency essential surgery should be a key priority for global child health agendas.
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| 2. |
- Amer, Ayad, 1980-
(författare)
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Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Several pathogenic Gram-negative bacteria invest in sophisticated type III secretion systems (T3SS) to incapacitate their eukaryotic hosts. T3SSs can secrete protein cargo outside the bacterial cell and also target many of them into the eukaryotic cell interior. Internalized proteins promote bacterial colonization, survival and transmission, and can often cause severe disease. An example is the Ysc-Yop T3SS apparatus assembled by pathogenic Yersinia spp. A correctly assembled Ysc-Yop T3SS spans the Yersinia envelope and also protrudes from the bacterial surface. Upon host cell contact, this system is competent to secrete hydrophobic translocators that form a translocon pore in the host cell membrane to complete the delivery channel bridging both bacterial and host cells. Newly synthesized effector Yops may pass through this channel to gain entry into the host cell cytosol.As type III secretion (T3S) substrates function sequentially during infection, it is hypothesized that substrate export is temporally controlled to ensure that those required first are prioritized for secretion. On this basis three functional groups are classified as early (i.e. structural components), middle (i.e. translocators) and late (i.e. effectors). Factors considered to orchestrate the T3S of substrates are many, including the intrinsic substrate secretion signal sequences, customized chaperones, and recognition/sorting platforms at the base of the assembled T3SS. Investigating the interplay between these elements is critical for a better understanding of the molecular mechanisms governing export control during Yersinia T3S.To examine the composition of the N-terminal T3S signals of the YscX early substrate and the YopD middle substrate, these segments were altered by mutagenesis and the modified substrates analyzed for their T3S. Translational fusions between these signals and a signalless β-Lactamase were used to determine their optimal length required for efficient T3S. This revealed that YscX and YopD export is most efficiently supported by their first 15 N-terminal residues. At least for YopD, this is a peptide signal and not base upon information in the mRNA sequence. Moreover, features within and upstream of this segment contribute to their translational control. In parallel, bacteria were engineered to produce substrate chimeras where the N-terminal segments were exchanged between substrates of different classes in an effort to examine the temporal dynamics of T3S. In several cases, Yersinia producing chimeric substrates were defective in T3S activity, which could be a consequence of disturbing a pre-existing hierarchal secretion mechanism.YopN and TyeA regulatory molecules can be naturally produced as a 42 kDa YopN-TyeA hybrid, via a +1 frame shift event somewhere at the 5’-end of yopN. To study this event, Yersinia were engineered to artificially produce this hybrid, and these maintained in vitro T3S control of both middle and late substrates. However, modestly diminished directed targeting of effectors into eukaryotic cells correlated to virulence attenuation in vivo. Upon further investigation, a YopN C-terminal segment encompassing residues 278 to 287 was probably responsible, as this region is critical for YopN to control T3S, via enabling a specific interaction with TyeA.Investigated herein were molecular mechanisms to orchestrate substrate export by the T3SS of Yersinia. While N-terminal secretion signals may contribute to specific substrate order, the YopN and TyeA regulatory molecules do not appear to distinguish between the different substrate classes.
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| 3. |
- Amer, Ayad, 1980-, et al.
(författare)
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Functional consequences of site-directed mutagenesis in the C-terminus of YopN, a Yersinia pseudotuberculosis regulator of Yop secretion
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Pathogenic Yersinia spp. utilizes the Ysc-Yop type III secretion system to targetYop effector proteins into the cytosol of host immune cells. Internalizedeffectors alter specific signaling pathways to neutralize immune cell-dependentphagocytosis, killing and pro-inflammatory responsiveness. This enablesextracellular bacterial multiplication and survival in immune tissue. Central tothe temporal control of Yop type III secretion is the regulator YopN. Incomplex with TyeA, YopN acts to plug the inner face of the type III secretionchannel, denying entry to other Yop substrates until after YopN has beensecreted. A +1 frameshift event in the 3-prime end of yopN results in thesynthesis of a singular secreted YopN-TyeA polypeptide chimera that retainssome regulatory function. As the C-terminal coding sequence of YopN in thishybrid product differs greatly from native sequence, we used site-directedmutagenesis to determine the functional significance of this segment. YopNtruncated at residue 287 or containing a shuffled sequence covering 288 to 293retains full function both in vitro and in vivo. Thus, the extreme C-terminus isapparently superfluous to YopN function. In contrast, a YopN varianttruncated after residue 278 was completely unstable, and these bacteria hadlost all control of T3S activity, and failed to defend against immune cell killing.Interestingly, inclusion of a shuffled sequence from residues 279 to 287recovered some T3S control over function. Hence, the YopN segmentencompassing 279 to 287 is essential for full function, although the exact aminoacid sequence is less important.
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| 4. |
- Amer, Ayad, 1980-, et al.
(författare)
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Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis
- 2013
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Ingår i: PLOS ONE. - San Francisco : Public Library of Science. - 1932-6203. ; 8:10
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Tidskriftsartikel (refereegranskat)abstract
- Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.
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| 5. |
- Amer, Ayad, et al.
(författare)
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Impact of the N-terminal secretor domain on YopD translocator function in Yersinia pseudotuberculosis type III secretion
- 2011
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 193:23, s. 6683-6700
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Tidskriftsartikel (refereegranskat)abstract
- Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5′ end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.
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| 6. |
- Amer, Ayad, 1980-, et al.
(författare)
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Yersinia pseudotuberculosis type III secretion is reliant upon an authentic N‐terminal YscX secretor domain
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Certain Gram‐negative bacteria use type III secretion systems to deliver effectorproteins into eukaryotic cells, serving either parasitic or mutualistic roles inside the hostcell. About 25 structural proteins are needed to assemble and deliver effector proteins.Collections of these proteins are quite well characterized, although the function ofsome continues to remain obscure. This is true for the Yersinia Ysc‐Yop systemcomponents YscX, a secreted substrate and YscY, its cognate non‐secreted chaperone.Despite recent evidence suggesting that they might coordinate Yop substrate secretion,YscX and YscY remain poorly characterized. To further investigate the function of theseproteins in the enteropathogen Y. pseudotuberculosis, we explored correlationsbetween the YscX N‐terminal segment, YscX secretion, as well as the secretion of otherYops. Analysis of a series of chimeric substrates in which the extreme YscX N‐terminushad been exchanged with equivalent functional secretion signals of other Ysc‐Yopsubstrates revealed that this segment contains non‐redundant information needed forYscX function, which includes permitting surface polymerization of the YscF needle andYops secretion. Further, in cis deletion of the YscX N‐terminus and ectopic expression ofepitope tagged YscX variants again correlated stable YscX production but not secretionto the type III secretion of Yops. Despite this, the first 5 codons were determined toconstitute a minimal signal capable of promoting secretion of the signalless ‐lactamasereporter. Hence, YscX does contain a fully equipped N‐terminal secretor domain topromote secretion of self. Nevertheless, the primary role of this N‐terminal segmentmust be to assemble an operational secretion system, and this occurs independently ofYscX secretion.
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| 7. |
- Amer, Ayad, et al.
(författare)
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YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis
- 2016
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Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media SA. - 2235-2988. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.
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| 8. |
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| 9. |
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| 10. |
- Costa, Tiago R. D., et al.
(författare)
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Active type III translocon assemblies that attenuate Yersinia virulence
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted a-helix motif at the C-terminus. Mutants YopDI262P and YopDK267P poorly localised Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopDA263P and YopDA270P had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. It is therefore probable that an active translocon makes a range of contributions during bacteria-host cell contact that extends beyond effector delivery per se.
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