The conserved mechanism of IcsA polar targeting among proteobacteria, characterization of the omptin family, and the roles and regulation of IcsP in Shigella flexneri

Shigella flexneri is a gram-negative intracellular pathogen that is a global health problem, causing severe bacillary dysentery. In the past 30 years, research has enabled us to have a better understanding of the molecular basis of Shigella pathogenesis. In this study, we further expand our knowledge of key determinants at molecular and cellular levels. This work is composed of four separate investigations; Firstly, we examined whether the elusive mechanism of protein targeting found in Shigella is conserved in three closely related gamma-proteobacterial families by using GFP fusions and microscopy. Our results indicate that the mechanism of protein localization to the bacterial pole was conserved in the species investigated. Secondly, we further characterized omptin proteins found in three pathogenic organisms: Shigella (IcsP), E. coli (OmpT), and Salmonella (PgtE). We addressed whether the LPS environment and/or the inherent amino acid differences in the surface loops and LPS binding motif affect the cleavage specificity of these proteases by introducing a known substrate into each of these strains and comparing cleavage patterns by western blot analysis. Our results indicate that the cleavage specificity of these omptins are likely due to the inherent amino acid differences in the surface loops and/or LPS binding region. Thirdly, we examined whether IcsP, an outer membrane protease in Shigella, promotes resistance to a cationic antimicrobial peptide, LL-37 by using minimum inhibitory concentration assays. Our results suggest that IcsP does not promote resistance to LL-37. Finally, we examined whether icsP was regulated by RyhB, a small regulatory RNA, by western blot analysis and beta-galactosidase assays. Our results suggest that RyhB does not down-regulate the expression of icsP in S. flexneri; Taken together, this study has improved our understanding of Shigella pathogenesis and omptin family proteases. Ultimately, the relevance of this study has potential use for the development of prevention and intervention strategies against specific bacterial infections and disease

Perception And Utilization Of Telehealth Services Among Home Health Care Agencies: A National Survey

Eun hae Kim Despite the widely known effectiveness of telehealth services in screening and treating both chronic disease and depression in older adults, their adoption among home health care agencies has been slow. Furthermore, there is a lack of empirical research on telehealth use, barriers, and facilitators of adoption in the home health sector. For these reasons, this study examined home health care staff perceptions and use of telehealth for chronic disease and depression care among older patients. Five hundred and sixteen staff from member home health care agencies of the National Association for Homecare and Hospice (NAHC) completed an online survey. The national survey comprised of 33 questions and was informed by the Unified Theory of Acceptance and Use of Technology Model (Venkatesh et al., 2003) and Bobni’ Innovation Culture paradigm. Twenty staff also participated in a 45-minute qualitative telephone interview. The qualitative interview questions focused on telehealth experience and perceived barriers and facilitators to telehealth adoption. Among HHC agencies that reported using telehealth, telephone (63%) and remote-monitoring devices (56%) were the most utilized telehealth technology. Telehealth services included monitoring of health services (64%), chronic disease management (58%), and patient health education (43%). Telehealth was the least used for depression counseling (15%). Overall, there was a positive perception towards telehealth for patient care. However, telehealth was perceived more positively for chronic disease management (90.7%) than for depression care (53%). A majority (74%) perceived themselves as having the knowledge necessary to use telehealth for chronically ill patients while only 32% did for depressed patients. Results suggest that although there is a positive perception towards telehealth for patient care, there are other factors (e.g., lack of resources and reimbursement, training and buy-in from staff or patients) that affect HHC agencies’ adoption and use of telehealth. Therefore, further education is needed to support telehealth use for depression care. Additionally, there needs to be a reimbursement for telehealth visits by HHC agencies, as well as policies and regulations that ensure the quality of care provided by telehealth services. Future studies may consider comparing existing telehealth programs and identifying policies and regulations that are supportive of such programs

Characterization of the omptin protease, OmpT, in Escherichia coli

Omptins are outer membrane proteases found in gram negative bacteria that cause diseases in humans, such as pathogenic Escherichia coli, Shigella flexneri, Salmonella typhimurium, and Yersinia pestis. Bacterial species that express omptins cause diseases such as highly fatal plague and severe diarrhea and dysentery. The genes that encode these proteases are ompT, icsP, pgtE, and pla, respectively. These proteases are highly related in structure and share approximately 50% sequence identity. In S. flexneri, IcsP has been shown to cleave a key virulence determinant, IcsA (Egile et al., 1997). IcsA recruits host actin and allows for intracellular movement within host cells (Steinhauer et al., 1999). In S. typhimurium, PgtE has been shown to cleave a human a-helical cationic antimicrobial peptide (CAMP), LL-37 (Guina et al., 2000). LL-37 is a major component of the innate immune defense system (Zasloff, 1992). It functions by permeabilizing the bacterial membrane, which ultimately results in bacterial cell lysis. PgtE cleaves LL-37, thereby protecting Salmonella against the bactericidal effects of CAMPs. In E. coli, OmpT has been shown to cleave protamine, an antimicrobial peptide that acts on the bacterial membrane and causes problems in cellular energy transduction and nutrient accumulation (Aspedon and Groisman, 1996) (Figure 2). Like PgtE, OmpT circumvents the immune defense by cleaving protamine into smaller fragments

Modifying the amino acid sequence in the surface-exposed loops of the omptin family of proteins to determine their effect on function

The omptin family of proteins consists of proteases which lie in the outer membrane of some gram-negative, pathogenic bacteria such as Escherichia coli (OmpT), Shigella flexneri (IcsP), Salmonella typhimurium (PgtE), and Yersinia pestis (Pla). These proteases are highly conserved, sharing approximately 50% sequence identity and a β-barrel shape (fig. 1D). The differences in the structure of these four proteins are in the surface-exposed loop region surrounding the active site, but not in the active site itself [4]. These proteases are important for the virulence of many bacteria. For example, OmpT of E. coli cleaves an antimicrobial peptide secreted by epithelial cells of the urinary tract [5]; IcsP of S. flexneri regulates IcsA, which uses the host’s actin to allow motility of the bacterium [6]; PgtE of S. typhimurium helps the bacterium evade the immune system by cleaving the α-helical cationic antimicrobial peptides [1]; and, Pla of Y. pestis enhances bacterial migration through tissue barriers by cleaving plasminogen [4]. Previous work [2] has shown that the omptin proteins of E. coli and S. typhimurium do not cleave IcsA in the same manner as IcsP in S. flexneri. Differences in the cleavage of IcsA may be due to the differences in surface-exposed loops of the protease or in its LPS binding motif [2]. Determining whether the surface-exposed loops of a protein affects its function could lead to a better understanding of this protein’s function and how it has evolved to serve different functions in different bacterial pathogens

Validation of the Dimensions of Anger Reactions Scale (the DAR-5) in non-clinical South Korean adults

Background Posttraumatic anger is a commonly reported emotion among people who have experienced traumatic events. The current study aimed to demonstrate the reliability and validity of the South Korean version of the DAR-5 (DAR-5-K). The DAR-5 is a single scale with 5 items which measures posttraumatic anger. The DAR-5 is composed of five items that measure anger frequency, intensity, duration, aggression, and its interference with social relations. Methods Data were collected from 814 South Korean adults who had experienced traumatic events and participated in the study and analyzed via the combination of exploratory factor analysis (n = 405) and confirmatory factor analysis (n = 409). Results Results supported the one-factor structure, as reported in previous validation studies. The scale demonstrated robust internal reliability and concurrent validity with measures of posttraumatic stress disorder (PTSD) symptoms, depression, anxiety, and self-esteem. The DAR-5 cut-off score of 12 that was established in the original validation study successfully differentiated high from low scorers with regard to PTSD symptoms, depression, anxiety, and self-esteem. Conclusion The results confirm that the DAR-5-K is a brief and psychometrically robust measure of anger that can be used to examine South Korean adults who have experienced traumatic events