The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 codes for two interacting DNA binding proteins, Cra and KdpE, that coregulate expression of the locus of enterocyte effacement ...(LEE) genes in a metabolite-dependent manner. Cra is a transcription factor that uses fluctuations in the concentration of carbon metabolism intermediates to positively regulate virulence of EHEC. KdpE is a response regulator that activates the transcription of homeostasis genes in response to salt-induced osmolarity and virulence genes in response to changes in metabolite concentrations. Here, we probed the transcriptional profiles of the Δcra, ΔkdpE, and Δcra ΔkdpE mutant strains and show that Cra and KdpE share several targets besides the LEE, but both Cra and KdpE also have independent targets. Several genes within O-islands (genomic islands present in EHEC but absent from E. coli K-12), such as Z0639, Z0640, Z3388, Z4267, and espFu (encoding an effector necessary for formation of attaching and effacing lesions on epithelial cells), were directly regulated by both Cra and KdpE, while Z2077 was only regulated by Cra. These studies identified and confirmed new direct targets for Cra and KdpE that included putative virulence factors as well as characterized virulence factors, such as EspFu and EspG. These results map out the role of the two interacting regulators, Cra and KdpE, in EHEC pathogenesis and global gene regulation.
Gastrointestinal (GI) bacteria sense diverse environmental signals as cues for differential gene regulation and niche adaptation. Pathogens such as enterohemorrhagic Escherichia coli (EHEC), which ...causes bloody diarrhea, use these signals for the temporal and energy-efficient regulation of their virulence factors. One of the main virulence strategies employed by EHEC is the formation of attaching and effacing (AE) lesions on enterocytes. Most of the genes necessary for the formation of these lesions are grouped within a pathogenicity island, the locus of enterocyte effacement (LEE), whose expression requires the LEE-encoded regulator Ler. Here we show that growth of EHEC in glycolytic environments inhibits the expression of ler and consequently all other LEE genes. Conversely, growth within a gluconeogenic environment activates expression of these genes. This sugar-dependent regulation is achieved through two transcription factors: KdpE and Cra. Both Cra and KdpE directly bind to the ler promoter, and Cra's affinity to this promoter is catabolite dependent. Moreover, we show that the Cra and KdpE proteins interact in vitro and that KdpE's ability to bind DNA is enhanced by the presence of Cra. Cra is important for AE lesion formation, and KdpE contributes to this Cra-dependent regulation. The deletion of cra and kdpE resulted in the ablation of AE lesions. One of the many challenges that bacteria face within the GI tract is to successfully compete for carbon sources. Linking carbon metabolism to the precise coordination of virulence expression is a key step in the adaptation of pathogens to the GI environment. IMPORTANCE An appropriate and prompt response to environmental cues is crucial for bacterial survival. Cra and KdpE are two proteins found in both nonpathogenic and pathogenic bacteria that regulate genes in response to differences in metabolite concentration. In this work, we show that, in the deadly pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7, which causes bloody diarrhea, these two proteins influence important virulence traits. We also propose that their control of one or more of these virulence traits is due to the direct interaction of the Cra and KdpE proteins with each other, as well as with their DNA targets. This work shows how EHEC coopts established mechanisms for sensing the metabolites and stress cues in the environment, to induce virulence factors in a temporal and energy-efficient manner, culminating in disease. Understanding how pathogens commandeer nonpathogenic systems can help us develop measures to control them.
Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in ...combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.
The histidine sensor kinase (HK) QseC senses autoinducer 3 (AI-3) and the adrenergic hormones epinephrine and norepinephrine. Upon sensing these signals, QseC acts through three response regulators ...(RRs) to regulate the expression of virulence genes in enterohemorrhagic
(EHEC). The QseB, QseF, and KdpE RRs that are phosphorylated by QseC constitute a tripartite signaling cascade having different and overlapping targets, including flagella and motility, the type three secretion system encoded by the locus of enterocyte effacement (LEE), and Shiga toxin. We modeled the tertiary structure of QseC's periplasmic sensing domain and aligned the sequences from 12 different species to identify the most conserved amino acids. We selected eight amino acids conserved in all of these QseC homologues. The corresponding QseC site-directed mutants were expressed and still able to autophosphorylate; however, four mutants demonstrated an increased basal level of phosphorylation. These mutants have differential flagellar, motility, LEE, and Shiga toxin expression phenotypes. We selected four mutants for more in-depth analyses and found that they differed in their ability to phosphorylate QseB, KdpE, and QseF. This suggests that these mutations in the periplasmic sensing domain affected the region downstream of the QseC signaling cascade and therefore can influence which pathway QseC regulates.
In the foodborne pathogen EHEC, QseC senses AI-3, epinephrine, and norepinephrine, increases its autophosphorylation, and then transfers its phosphate to three RRs: QseB, QseF, and KdpE. QseB controls expression of flagella and motility, KdpE controls expression of the LEE region, and QseF controls the expression of Shiga toxin. This tripartite signaling pathway must be tightly controlled, given that flagella and the type three secretion system (T3SS) are energetically expensive appendages and Shiga toxin expression leads to bacterial cell lysis. Our data suggest that mutations in the periplasmic sensing loop of QseC differentially affect the expression of the three arms of this signaling cascade. This suggests that these point mutations may change QseC's phosphotransfer preferences for its RRs.
This chapter contains sections titled:
Introduction
Two‐Component Systems (TCSs) as Potential Anti‐Infective Targets
WalK/WalR and MtrB/MtrA: Case Studies of Essential TCSs as Drug Targets
Targeting ...Nonessential TCS
Non‐TCSs Targeting Biofilm Formation and Quorum Sensing in Pseudomonas spp.
Conclusions
References
Pregnant women in settings with high HIV prevalence are at increased risk of HIV acquisition and subsequent vertical transmission. We implemented and evaluated a novel programme to provide ...pre-exposure prophylaxis (PrEP) in maternal and child health clinics in Kenya.
In collaboration with Kisumu County Department of Health, we integrated PrEP delivery within 16 maternal and child health clinics in Kisumu County (Kenya). Women and girls older than 15 years seeking maternal and child health services who tested HIV negative at that visit or within a month and were willing to receive PrEP counselling were interviewed to assess for HIV behavioural risk factors and offered PrEP. Correlates of PrEP initiation and continuation were assessed using Poisson regression in univariate and multivariate analyses. Potential correlates included in our analyses were age, marital status, marriage type, whether pregnant or post partum, gestational age (if pregnant), and HIV risk factors in the previous 6 months. Reasons for the decision to discontinue after having decided to initiate PrEP were evaluated. Women who initiated PrEP were followed up 1 month, 3 months, and 6 months after initiation.
Between Nov 20, 2017, and June 13, 2018, 9376 pregnant and post-partum women were assessed for behavioural risk factors and willingness to initiate PrEP. Overall, 2030 (21·7%) initiated PrEP, and 2027 had the status of their partner captured (153 79·3% of 193 women with partners living with HIV, 1178 37·2% of 3165 women with partners of unknown HIV status, and 696 11·6% of 5997 women with HIV-negative partners). Predictors of PrEP initiation in the multivariate analysis were: being younger than 24 years (adjusted prevalence ratio 1·14, 95% CI 1·02-1·28); having a partner living with HIV (6·96, 5·46-8·89) or of unknown HIV status (3·08, 2·50-3·81); gestational age of less than 26 weeks (1·22, 1·02-1·47); having been diagnosed or treated for a sexually transmitted infection (1·57, 1·20-2·06); having been forced to have sex (1·82, 1·38-2·42); having experienced intimate partner violence during the previous 6 months (1·65, 1·10-2·48); having shared needles while engaging in injection drug use (2·43, 1·69-3·50); and recurrent use of post-exposure prophylaxis (1·96, 1·36-2·82). Overall, 786 (38·7%) of 2030 women who initiated PrEP continued use after the first month, with 104 (68·0%) of 153 women who had a partner living with HIV continuing use. Having a partner living with HIV was the only predictor of PrEP continuation at 1 month in the multivariable model (1·98, 1·54-2·55). Frequent reasons for discontinuation were side effects and low HIV risk perception. No incident HIV infection was reported among women on PrEP.
Many women attending maternal and child health clinics had risk factors for HIV and elected to use PrEP, indicating that routinely accessed maternal and child health clinics can be an effective platform for PrEP delivery for young women. As PrEP awareness rises, PrEP provision in routine clinical settings such as maternal and child health facilities might contribute to decreased HIV incidence among young women.
US Department of State.
Abstract
Domestication of the helmeted guinea fowl (HGF; Numida meleagris) in Africa remains elusive. Here we report a high-quality de novo genome assembly for domestic HGF generated by long- and ...short-reads sequencing together with optical and chromatin interaction mapping. Using this assembly as the reference, we performed population genomic analyses for newly sequenced whole-genomes for 129 birds from Africa, Asia, and Europe, including domestic animals (n = 89), wild progenitors (n = 34), and their closely related wild species (n = 6). Our results reveal domestication of HGF in West Africa around 1,300–5,500 years ago. Scanning for selective signals characterized the functional genes in behavior and locomotion changes involved in domestication of HGF. The pleiotropy and linkage in genes affecting plumage color and fertility were revealed in the recent breeding of Italian domestic HGF. In addition to presenting a missing piece to the jigsaw puzzle of domestication in poultry, our study provides valuable genetic resources for researchers and breeders to improve production in this species.
This paper reports on the deposition of pure and 5 at% Al doped ZnO (AZO) prepared by sol-gel and applied to the substrates by spin-coating, and the role of annealing temperature on the crystallinity ...of these layers. It is found that both ZnO and AZO are largely amorphous when coated on glass compared to n-Si(lll), as substrates. On both substrates, X-ray diffraction (XRD) shows that the crystallinity improves as annealing temperature is raised from 200 to 600 degreesC with better crystallinity on Si substrates. The thickness of the films on substrates was determined as 120 nm by Rutherford backscattering spectroscopy (RBS). Specular ultra-violet visible (UV-vis) gives the direct transition optical band gaps (E sub(g)) for AZO as-deposited films are 2.60 and 3.35 eV while that of 600 degreesC annealed films are 3.00 and 3.60 eV. The E sub(g) calculated from diffuse reflectance spectroscopy (DRS) UV-vis are more diverse in ZnO- and AZO-Si than the ZnO- and AZO-glass samples, although in both sets the E sub(g) tend to converge after annealing 600 degreesC. The Raman spectra of samples show multiphonon processes of higher order from the AZO and substrates. It is found that residual stresses are related to E sub(2) Raman mode.
This paper reports on the deposition of pure and 5at% Al doped ZnO (AZO) prepared by sol–gel and applied to the substrates by spin-coating, and the role of annealing temperature on the crystallinity ...of these layers. It is found that both ZnO and AZO are largely amorphous when coated on glass compared to n-Si(111), as substrates. On both substrates, X-ray diffraction (XRD) shows that the crystallinity improves as annealing temperature is raised from 200 to 600°C with better crystallinity on Si substrates. The thickness of the films on substrates was determined as 120nm by Rutherford backscattering spectroscopy (RBS). Specular ultra-violet visible (UV–vis) gives the direct transition optical band gaps (Eg) for AZO as-deposited films are 2.60 and 3.35eV while that of 600°C annealed films are 3.00 and 3.60eV. The Eg calculated from diffuse reflectance spectroscopy (DRS) UV–vis are more diverse in ZnO- and AZO-Si than the ZnO- and AZO-glass samples, although in both sets the Eg tend to converge after annealing 600°C. The Raman spectra of samples show multiphonon processes of higher order from the AZO and substrates. It is found that residual stresses are related to E2 Raman mode.
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