Julie M. Silverman

A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria.

The functional spectrum of a secretion system is defined by its substrates. Here we analyzed the secretomes of Pseudomonas aeruginosa mutants altered in regulation of the Hcp Secretion Island-I-encoded type VI secretion system (H1-T6SS). We identified three substrates of this system, proteins Tse1-3 (type six exported 1-3), which are coregulated with the secretory apparatus and secreted under tight posttranslational control. The Tse2 protein was found to be the toxin component of a toxin-immunity system and to arrest the growth of prokaryotic and eukaryotic cells when expressed intracellularly. In contrast, secreted Tse2 had no effect on eukaryotic cells; however, it provided a major growth advantage for P. aeruginosa strains, relative to those lacking immunity, in a manner dependent on cell contact and the H1-T6SS. This demonstration that the T6SS targets a toxin to bacteria helps reconcile the structural and evolutionary relationship between the T6SS and the bacteriophage tail and spike.

Structure and Regulation of the Type VI Secretion System

The type VI secretion system (T6SS) is a complex and widespread gram-negative bacterial export pathway with the capacity to translocate protein effectors into a diversity of target cell types. Current structural models of the T6SS indicate that the apparatus is composed of at least two complexes, a dynamic bacteriophage-like structure and a cell-envelope-spanning membrane-associated assembly. How these complexes interact to promote effector secretion and cell targeting remains a major question in the field. As a contact-dependent pathway with specific cellular targets, the T6SS is subject to tight regulation. Thus, the identification of regulatory elements that control T6S expression continues to shape our understanding of the environmental circumstances relevant to its function. This review discusses recent progress toward characterizing T6S structure and regulation.

Haemolysin Coregulated Protein Is an Exported Receptor and Chaperone of Type VI Secretion Substrates

Secretion systems require high-fidelity mechanisms to discriminate substrates among the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system (T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcp-effector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways.

Separate inputs modulate phosphorylation‐dependent and ‐independent type VI secretion activation

Productive intercellular delivery of cargo by secretory systems requires exquisite temporal and spatial choreography. Our laboratory has demonstrated that the haemolysin co‐regulated secretion island I (HSI‐I)‐encoded type VI secretion system (H1‐T6SS) of Pseudomonas aeruginosa transfers effector proteins to other bacterial cells. The activity of these effectors requires cell contact‐dependent delivery by the secretion apparatus, and thus their export is highly repressed under planktonic growth conditions. Here we define regulatory pathways that orchestrate efficient secretion by this system. We identified a T6S‐associated protein, TagF, as a posttranslational repressor of the H1‐T6SS. Strains activated by TagF derepression or stimulated through a previously identified threonine phosphorylation pathway (TPP) share the property of secretory ATPase recruitment to the T6S apparatus, yet display different effector output levels and genetic requirements for their export. We also found that these two pathways respond to distinct stimuli; we identified surface growth as a physiological cue that activates the H1‐T6SS exclusively through the TPP. Coordination of posttranslational triggering with cell contact‐promoting growth conditions provides a mechanism for the T6SS to avoid wasteful release of effectors.

An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa

Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions.

A Minimalist Substrate for Enzymatic Peptide and Protein Conjugation

Recently a number of nonnatural prenyl groups containing alkynes and azides have been developed as handles to perform click chemistry on proteins and peptides ending in the sequence “CAAX”, where C is a cysteine that becomes alkylated, A is an aliphatic amino acid and X is any amino acid. When such molecules are modified, a tag containing a prenyl analogue and the “CAAX box” sequence remains. Here we report the synthesis of an alkyne‐containing substrate comprised of only nine nonhydrogen atoms. This substrate was synthesized in six steps from 3‐methylbut‐2‐en‐1‐ol and has been enzymatically incorporated into both proteins and peptides by using protein farnesyltransferase. After prenylation the final three amino acids required for enzymatic recognition can be removed by using carboxypeptidase Y, leaving a single residue (the cysteine from the “CAAX box”) and the prenyl analogue as the only modifications. We also demonstrate that this small tag minimizes the impact of the modification on the solubility of the targeted protein. Hence, this new approach should be useful for applications in which the presence of a large tag hinders the modified protein’s solubility, reactivity, or utility.

Peer Support for Achieving Independence in Diabetes (Peer-AID): Design, methods and baseline characteristics of a randomized controlled trial of community health worker assisted diabetes self-management support ☆

BACKGROUND & OBJECTIVES Community health workers (CHWs) may be an important mechanism to provide diabetes self-management to disadvantaged populations. We describe the design and baseline results of a trial evaluating a home-based CHW intervention. METHODS & RESEARCH DESIGN Peer Support for Achieving Independence in Diabetes (Peer-AID) is a randomized, controlled trial evaluating a home-based CHW-delivered diabetes self-management intervention versus usual care. The study recruited participants from 3 health systems. Change in A1c measured at 12 months is the primary outcome. Changes in blood pressure, lipids, health care utilization, health-related quality of life, self-efficacy and diabetes self-management behaviors at 12 months are secondary outcomes. RESULTS A total of 1438 patients were identified by a medical record review as potentially eligible, 445 patients were screened by telephone for eligibility and 287 were randomized. Groups were comparable at baseline on socio-demographic and clinical characteristics. All participants were low-income and were from diverse racial and ethnic backgrounds. The mean A1c was 8.9%, mean BMI was above the obese range, and non-adherence to diabetes medications was high. The cohort had high rates of co-morbid disease and low self-reported health status. Although one-third reported no health insurance, the mean number of visits to a physician in the past year was 5.7. Trial results are pending. CONCLUSIONS Peer-AID recruited and enrolled a diverse group of low income participants with poorly controlled type 2 diabetes and delivered a home-based diabetes self-management program. If effective, replication of the Peer-AID intervention in community based settings could contribute to improved control of diabetes in vulnerable populations.

Methods Employed to Assess Weight Loss in Older Adults by Means of Electronic Medical Records: A Systematic Review

ABSTRACT Electronic medical records (EMRs) can be used to identify and categorize weight loss in older adults, but research has not scrutinized methods for doing so. Through a modified PRISMA protocol, we systematically reviewed published methods for quantifying weight change from EMRs. Articles (all available through July 2016) were identified through PubMed and SCOPUS searches, screened, and evaluated. We abstracted relevant data and tabulated the methods to assess weight change. The 13 selected articles showed little consistency in the approach to key methodological issues: 1) time ranges assessed; 2) removal of spurious values; 3) metrics to quantify weight change; 4) number of measures needed to estimate change; 5) threshold for significant weight change; and 6) relation to ideal weight. There was essentially no consensus around how to identify and categorize weight loss. Further investigation is needed to establish scientifically validated and clinically useful algorithms, accounting for the six issues above.

The Value of Community Health Workers in Diabetes Management in Low-Income Populations: A Qualitative Study

To describe community health workers (CHWs) roles in a diabetes self-management intervention. Retrospective qualitative inductive analysis of open text home visit encounter form from Peer Support for Achieving Independence in Diabetes (Peer AID), a randomized controlled trial in which low-income individuals with poorly controlled diabetes received either CHW home visits or usual care. Following visits, CHWs completed encounter forms documenting the health goal of the visit, the self-management strategies discussed and participant concerns. 634 encounter reports were completed for the 145 intervention participants. CHW notes revealed three main obstacles to optimal disease control: gaps in diabetes knowledge and self-management skills; socioeconomic conditions; and the complexity of the healthcare system. CHWs helped participants overcome these obstacles through extensive, hands-on education, connecting participants to community resources, and assistance navigating the medical system. In addition, the CHWs offered uncomplicated accessibility and availability to their clients. CHWs can be a valuable asset for low-income patients with chronic health conditions who may require more support than what can provided in a typical primary care visit.

Veteran peer Coaches Optimizing and Advancing Cardiac Health (Vet-COACH); design and rationale for a randomized controlled trial of peer support among Veterans with poorly controlled hypertension and other CVD risks

BACKGROUND Peer support can improve health for patients with chronic conditions; however, evidence for disease prevention is less clear and peer recruitment strategies are not well described. This paper describes a study protocol to evaluate a peer support intervention to improve hypertension control and reduce cardiovascular disease (CVD) risk. METHODS & RESEARCH DESIGN Target enrollment for this two-site study is n = 400. Eligibility criteria include Veterans enrolled in Veterans Health Administration (VHA) primary care with poorly controlled hypertension and one other cardiovascular disease risk (smoking, overweight/obesity, or hyperlipidemia) who live in census tracts with high rates of hypertension. Enrolled participants are randomized to a home-based peer delivered self-management intervention (5 home visits and 5 phone calls with a peer health coach) versus usual care. The primary outcome is a change in systolic blood pressure (SBP) and secondary outcomes include change in CVD risk and health care use. RESULTS Trial results are pending and participant enrollment is ongoing. We recruited peer coaches from Veterans who lived in census tracks with the highest rates of hypertension. To recruit Veteran peer coaches, we asked primary care providers (n = 41) and team nurses (n = 35) to nominate patients who they thought would be a good fit for the peer coach position (based on successful self-management and health care navigation) (n = 73 nominated from 964 patients). We interviewed 12 Veterans and trained 5 peer coaches. CONCLUSIONS Results of this trial will inform peer support programs targeted to provide community-based delivery of prevention services to patients in high-risk areas. TRIAL REGISTRATION Clinicaltrial.gov identifier NCT02697422 TRIAL STATUS: Enrollment for the randomized trial phase began in September 2017 and will be complete September 2019.