Many Gram-negative bacteria use type III secretion systems (T3SSs) to inject virulence effector proteins into eukaryotic cells. The T3SS apparatus (T3SA) is structurally conserved among diverse ...bacterial pathogens and consists of a cytoplasmic sorting platform, an envelope-spanning basal body, and an extracellular needle with tip complex. The sorting platform is essential for effector recognition and powering secretion. Studies using bacterial “minicells” have revealed an unprecedented level of structural detail of the sorting platform; however, many of the structure-function relationships within this complex remain enigmatic. Here, we report on improved cryo-electron tomographic approaches to enhance the resolution of the Shigella T3SA sorting platform (at ≤2 nm resolution) done in concert with biochemical and genetic methods to define the sorting platform interactome and interactions with the T3SA inner membrane ring (IR). We observed that the sorting platform consists of “pods” with 6-fold symmetry that interact with the Spa47 ATPase via radial extensions comprising MxiN. Most importantly, MxiK maintained an interaction with the IR via specific interactions with the cytoplasmic domain of the IR protein MxiG (MxiGC), which is a noncanonical forkhead-associated domain, and MxiK has an elongated structure that interacts with the IR via MxiGC. T4 lysozyme-mediated insertional mutagenesis of MxiK revealed its orientation within the sorting platform and enabled disruption of interactions with its binding partners, which abolished sorting platform assembly. Finally, a comparison with the homologous interactions in the Salmonella T3SS sorting platform revealed clear differences in their IR-sorting platform interfaces that have possible mechanistic implications.
Abstract Purpose To examine the event-level association between alcohol consumption and the likelihood of unprotected sex among college-age young adults considering contextual factors of partner type ...and amount of alcohol consumed. Methods A 30-day, Web-based, structured daily diary was used to collect daily reports of sexual behaviors and alcohol use from 116 sexually active young adults, yielding 2,764 diary records. Each day we assessed the prior evening's behavior regarding alcohol consumption, opportunities for sex, sexual intercourse, condom use, and contextual factors including type of sexual partner. Results Based on multilevel models, drinking proximal to events of sexual intercourse increased the likelihood of unprotected sex with casual but not steady partners. For women there was a positive association between number of drinks and a greater likelihood of unprotected sex with casual partners but a negative association with steady partners. Drinking during situations involving opportunities for sex with casual partners increased the likelihood of sex. For women especially, drinking more increased the likelihood of sex occurring regardless of partner type. Conclusions Failure to assess the contextual determinants of the alcohol—unprotected sex association may result in underestimates of the magnitude of this association. These data highlight an important area for intervention with young adults: reducing alcohol-involved sexual risk behavior with casual partners, especially among women.
Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to ...cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 Å and 2.8 Å limiting resolution, respectively. These newly identified domains are composed of extended-length (114 Å in IpaB and 71 Å in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.
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► Truncation at the hydrophobic domain of first translocators IpaB and SipB yielded a soluble N-terminal core. ► A coiled-coil motif highlights the N-terminus of the translocator proteins IpaB and SipB. ► Intramolecular coiled coils are integral structural scaffolds in T3SSs. ► First translocator coiled coils share strong homology with the receptor domain of pore-forming colicins. ► This work represents an important starting point for subsequent structure–function analysis of first translocators.
Background: In some jurisdictions, persons who are convicted of driving under the influence of alcohol (DUI) are allowed to serve some portion of their prison sentence under home confinement as part ...of Intensive Supervision Programs (ISPs) which include pre-release psycho-education and close post-release supervision. Objectives: Test the hypothesis that persons convicted of DUI offenses who have spent some portion of their sentence under home confinement, as compared to a historical comparison group, will exhibit a relatively low re-conviction rate. Methods: Using administrative data for 1,410 repeat DUI offenders (302 members of the historical comparison group, 948 ISP members, and 160 persons who appear in both groups at different points in time), with a follow-up period of up to 3 years and 10 months, a marginal Cox model was employed to compare conviction rates of persons who experienced intensive supervision and home confinement with historical comparison group members. Results: Persons with ISP + home confinement experience a re-conviction rate that is less than half that observed in the comparison group. Age, ethnicity (white vs. non-white), and gender are also significant predictors of re-conviction. Conclusion: Home confinement, in conjunction with psycho-education and other program elements, is one means of reducing the costs of incarceration. The results of this study suggest that, in addition to cost savings, states may realize a public safety benefit in the form of a reduction in DUI offense rates.
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Dostopno za:
DOBA, FSPLJ, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to ...promote entry and create an important intracellular niche. The injectisome's cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body
MxiK and connect to the Spa47 ATPase
MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33
). The structure of Spa33
is known, but the precise makeup and structure of the pods
remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs
the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the
and
T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.
One objective of translational science is to identify elements of human immunodeficiency virus (HIV) risk-reduction interventions that have been shown to be effective and find new ways of delivering ...these interventions to the community to ensure that they reach the widest possible audience of at-risk individuals. The current study reports the development and evaluation of a computer-delivered, theory-based, individually tailored HIV risk-reduction intervention.
This study evaluated the effectiveness of a custom computerized HIV/AIDS risk reduction intervention at increasing HIV/AIDS preventive behaviors in a randomized trial with 157 college students. The intervention content and delivery were based on the Information-Motivation-Behavioral Skills Model of Health Behavior Change and used Motivational Interviewing techniques. Participants completed a baseline assessment of HIV prevention information, motivation, behavioral skills and behavior, attended two brief computer-delivered intervention sessions, and completed a follow-up assessment.
As compared to the control group (a nutrition education tutorial), participants who interacted with the computer-delivered HIV/AIDS risk reduction intervention exhibited a significant increase in risk reduction behavior. Specifically, participants reported a greater frequency of keeping condoms available and displayed greater condom-related knowledge at a four-week follow-up session; among sexually active participants, there was a significant increase in self-reported condom use.
Delivery of brief individually tailored HIV/AIDS risk reduction interventions via computer may be an effective HIV/AIDS prevention approach for adolescents. More research is needed to further support the effectiveness of this type of intervention and determine the generalizability of these findings to economically and educationally disadvantaged adolescents.
A motivational/individual differences model of infidelity is proposed in the course of empirically evaluating the traditional dichotomy between emotional and sexual motives. A scale assessing ...motivations for infidelity was developed and administered to 432 college students, 120 of whom reported past dating infidelity. Four motivations were identified and were associated as predicted with Big Five and other trait constructs; Sex was predicted by male gender, lower age, and unrestricted sociosexual orientation (SO); Dissatisfaction was predicted by female gender and Extraversion; Neglect was predicted by Neuroticism; and Anger by Neuroticism and low Agreeableness. As predicted, a two-factor model provides a poorer fit with the data than a multi-factor model. Unrestricted SO partially mediates the gender difference in endorsement of a sex motive for infidelity.
Bacterial type III secretion systems (T3SS) are used to inject proteins into mammalian cells to subvert cellular functions. The Shigella T3SS apparatus (T3SA) is comprised of a basal body, ...cytoplasmic sorting platform and exposed needle with needle “tip complex” (TC). TC maturation occurs when the translocator protein IpaB is recruited to the needle tip where both IpaD and IpaB control secretion induction. IpaB insertion into the host membrane is the first step of translocon pore formation and secretion induction. We employed disruptive insertional mutagenesis, using bacteriophage T4 lysozyme (T4L), within predicted IpaB loops to show how topological features affect TC functions (secretion control, translocon formation and effector secretion). Insertions within the N‐terminal half of IpaB were most likely to result in a loss of steady‐state secretion control, however, all but the two that were not recognized by the T3SA retained nearly wild‐type hemolysis (translocon formation) and invasiveness levels (effector secretion). In contrast, all but one insertion in the C‐terminal half of IpaB maintained secretion control but were impaired for hemolysis and invasion. These nature of the data suggest the latter mutants are defective in a post‐secretion event, most likely due to impaired interactions with the second translocator protein IpaC. Intriguingly, only two insertion mutants displayed readily detectable T4L on the bacterial surface. The data create a picture in which the makeup and structure of a functional T3SA TC is highly amenable to physical perturbation, indicating that the tertiary structure of IpaB within the TC is more plastic than previously realized.
Numerous Gram-negative pathogens infect eukaryotes and use the type III secretion system (T3SS) to deliver effector proteins into host cells. One important T3SS feature is an extracellular needle ...with an associated tip complex responsible for assembly of a pore-forming translocon in the host cell membrane. Shigella spp. cause shigellosis, also called bacillary dysentery, and invade colonic epithelial cells via the T3SS. The tip complex of Shigella flexneri contains invasion plasmid antigen D (IpaD), which initially regulates secretion and provides a physical platform for the translocon pore. The tip complex represents a promising therapeutic target for many important T3SS-containing pathogens. Here, in an effort to further elucidate its function, we created a panel of single-VH domain antibodies (VHHs) that recognize distinct epitopes within IpaD. These VHHs recognized the in situ tip complex and modulated the infectious properties of Shigella. Moreover, structural elucidation of several IpaD–VHH complexes provided critical insights into tip complex formation and function. Of note, one VHH heterodimer could reduce Shigella hemolytic activity by >80%. Our observations along with previous findings support the hypothesis that the hydrophobic translocator (IpaB in Shigella) likely binds to a region within the tip protein that is structurally conserved across all T3SS-possessing pathogens, suggesting potential therapeutic avenues for managing infections by these pathogens.
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