Naturally Occurring Off-Switches for CRISPR-Cas9.

CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT.

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.

Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

Prevalence of Slam-dependent hemophilins in Gram-negative bacteria.

Iron acquisition systems are crucial for pathogen growth and survival in iron-limiting host environments. To overcome nutritional immunity, bacterial pathogens evolved to use diverse mechanisms to acquire iron. Here, we examine a heme acquisition system that utilizes hemophores called hemophilins which are also referred to as HphAs in several Gram-negative bacteria. In this study, we report three new HphA structures from Stenotrophomonas maltophilia, Vibrio harveyi, and Haemophilus parainfluenzae. Structural determination of HphAs revealed an N-terminal clamp-like domain that binds heme and a C-terminal eight-stranded ß-barrel domain that shares the same architecture as the Slam-dependent Neisserial surface lipoproteins. The genetic organization of HphAs consists of genes encoding a Slam homolog and a TonB-dependent receptor (TBDR). We investigated the Slam-HphA system in the native organism or the reconstituted system in Escherichia coli cells and found that the efficient secretion of HphA depends on Slam. The TBDR also played an important role in heme uptake and conferred specificity for its cognate HphA. Furthermore, bioinformatic analysis of HphA homologs revealed that HphAs are conserved in the alpha, beta, and gammaproteobacteria. Together, these results show that the Slam-dependent HphA-type hemophores are prevalent in Gram-negative bacteria and further expand the role of Slams in transporting soluble proteins. IMPORTANCE: This paper describes the structure and function of a family of Slam (Type IX secretion System) secreted hemophores that bacteria use to uptake heme (iron) while establishing an infection. Using structure-based bioinformatics analysis to define the diversity and prevalence of this heme acquisition pathway, we discovered that a large portion of gammaproteobacterial harbors this system. As organisms, including Acinetobacter baumannii, utilize this system to facilitate survival during host invasion, the identification of this heme acquisition system in bacteria species is valuable information and may represent a target for antimicrobials.

Test and Treat for Prediabetes: A Review of the Health Effects of Prediabetes and the Role of Screening and Prevention.

The term prediabetes describes blood glucose levels above the normal range but below the threshold to diagnose type 2 diabetes. Several population health initiatives encourage a test and treat approach for prediabetes. In this approach, screening and identification of individuals with prediabetes should be followed by prompt referral to structured lifestyle modification programs or pharmacologic interventions that have been shown to prevent or delay the progression to type 2 diabetes in clinical trials. Here we provide a critical review of evidence for this test and treat approach by examining health outcomes associated with prediabetes and the availability and effectiveness of lifestyle modification approaches that target prediabetes. We also describe current limitations to the reach and uptake of evidence-based treatment options for prediabetes. Finally, we highlight lessons learned from identifying and labeling other preconditions to consider challenges and opportunities that may arise with increasing awareness of prediabetes as part of routine preventive care.

A Culturally Adapted, Telehealth, Community Health Worker Intervention on Blood Pressure Control among South Asian Immigrants with Type II Diabetes: Results from the DREAM Atlanta Intervention.

BACKGROUND: South Asians face a high prevalence of type II diabetes (DMII) and comorbid hypertension (HTN). Community health worker (CHW) interventions have the potential to improve chronic disease outcomes, yet few have been tailored to South Asian populations in the United States. OBJECTIVE: To test the effectiveness of an evidence-based CHW-led and culturally-tailored HTN and DMII management program for South Asian adults with diabetes and comorbid uncontrolled HTN (systolic blood pressure (SBP) > 130 mmHg or diastolic blood pressure (DBP) > 80 mmHg). DESIGN: Randomized-controlled Trial. PARTICIPANTS: South Asian adults with DMII and comorbid HTN. INTERVENTION: The Diabetes Research, Education, and Action for Minorities (DREAM) Atlanta intervention was a CHW telehealth intervention designed to improve blood pressure (BP). The treatment group received five virtual group-based health education sessions, an action plan, and follow-up calls to assess goal setting activities. The control group received only the first session. Main Measures included: feasibility, improvement in BP control, and decreases in SBP, DBP, weight, and hemoglobin A1c (HbA1c). KEY RESULTS: A total of 190 South Asian adults were randomized (97 to the treatment group and 93 to the control group); 94% of treatment group participants completed all 5 telehealth sessions. At endpoint, BP control increased 33.7% (95% CI: 22.5, 44.9, p < 0.001) in the treatment group and 16.5% (95%: 6.2, 26.8, p = 0.003) in the control group; the adjusted intervention effect was 1.8 (95% CI: 1.0, 3.2, p = 0.055). Mean weight decreased by 4.8 pounds (95% CI: -8.2, -1.4, p = 0.006) in the treatment group, and the adjusted intervention effect was -5.2 (95% CI: -9.0, -1.4, p = 0.007. The intervention had an overall retention of 95%. CONCLUSIONS: A culturally-tailored, CHW-led telehealth intervention is feasible and can improve BP control among South Asian Americans with DMII. GOV REGISTRATION: NCT04263311.

Modulation of various host cellular machinery during COVID-19 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) emerged in December 2019, causing a range of respiratory infections from mild to severe. This resulted in the ongoing global COVID-19 pandemic, which has had a significant impact on public health. The World Health Organization declared COVID-19 as a global pandemic in March 2020. Viruses are intracellular pathogens that rely on the host's machinery to establish a successful infection. They exploit the gene expression machinery of host cells to facilitate their own replication. Gaining a better understanding of gene expression modulation in SARS-CoV2 is crucial for designing and developing effective antiviral strategies. Efforts are currently underway to understand the molecular-level interaction between the host and the pathogen. In this review, we describe how SARS-CoV2 infection modulates gene expression by interfering with cellular processes, including transcription, post-transcription, translation, post-translation, epigenetic modifications as well as processing and degradation pathways. Additionally, we emphasise the therapeutic implications of these findings in the development of new therapies to treat SARS-CoV2 infection.

A Comparison of Los Angeles County Health Survey Outcomes After Transitioning From Telephone to a Primarily Web-Based, Self-Administration Data Collection Mode.

CONTEXT: As response rates to health surveys conducted by telephone continue to fall while data collection costs rise, practitioners are increasingly transitioning to address-based sample (ABS) designs with a self-administered, mail contact data collection mode. OBJECTIVE: To compare differences in key health indicators produced from both the Adult Survey and Child Survey of the Los Angeles County Health Survey (LACHS), which transitioned from a telephone to primarily self-administered mode in the 2022-2023 administration. DESIGN: Weighted survey estimates from the 2015, 2018, and 2022-2023 LACHS administrations are compared to investigate differences that may be attributable to LACHS' recent mode transition. SETTING: All survey data were collected of residents of Los Angeles County between 2015 and 2023. MAIN OUTCOME MEASURES: Response rates and key health indicators produced from the Los Angeles County Health Survey. RESULTS: Introducing the self-administration mode led to modest response rate increases of approximately 10% to 16% for the Adult Survey and from 10% to 14% in the Child Survey. Key health indicator differences are mixed, but generally larger in magnitude across the mode transition, and also generally larger for the Adult Survey relative to the Child Survey. CONCLUSIONS: Transitioning a population health survey from a telephone mode to a primarily self-administration mode using an ABS design comes with tradeoffs. Increased response rates and a greater ability to target lower-level geographies and other population domains of interest may be offset by mode effects that cannot be compensated for by weighting adjustments.

Cardiometabolic Risk in Asian Americans by Social Determinants of Health: Serial Cross-sectional Analyses of the NHIS, 1999-2003 to 2014-2018.

BACKGROUND: Diabetes and hypertension are common in Asian Americans and vary by subgroup. There may be further variation by social determinants of health (SDOHs), but few studies have examined this previously. OBJECTIVE: To examine the associations of SDOHs and diabetes and hypertension within and across Asian subgroups in the USA DESIGN: Series cross-sectional analyses SETTING: National Health Interview Surveys (NHIS) from 1999 to 2018 PARTICIPANTS: Asian-American adults (Chinese, Filipino, Asian Indian, and Other Asian [Korean, Vietnamese, Japanese, and other]) MEASUREMENTS: Self-reported diabetes and hypertension prevalence in pooled 5-year increments over 1999-2018 and multivariable regression models to assess the adjusted prevalence of diabetes or hypertension by poverty, marital status, education, and years in the USA, adjusting for age, sex, BMI, and health insurance status RESULTS: From 1999-2003 to 2014-2018, the age- and sex-adjusted prevalence of diabetes increased for Other Asians (absolute change: 4.6%) but not for other subgroups; age- and sex-adjusted hypertension prevalence significantly increased for Asian Indians and Other Asians (absolute change: 5-7.5%). For Filipinos, high school education or less was associated with an increase in diabetes prevalence over time (difference from 1999-2003 to 2014-2018: +6.0 (95% CI: 2.0-10.0)), while for Asian Indians, college education or higher was associated with an increase in diabetes prevalence for the same period (difference: +2.7 (95% CI: 0.01-5.4). Differences over the 2 time periods (1999-2003 and 2014-2018) show that Filipino and Other Asians, who lived in the USA for ≥10 years, increased in diabetes prevalence. Similar variations in associations of SDOHs by Asian subgroup were seen for hypertension. LIMITATIONS: Self-reported primary outcomes and multi-year data were pooled due to small sample sizes. CONCLUSIONS: The influence of SDOHs on cardiometabolic risk is not uniform among Asian Americans, implying tailored strategies may be needed for different population subgroups. PRIMARY FUNDING SOURCE: NIH.

Actinobacillus utilizes a binding protein-dependent ABC transporter to acquire the active form of vitamin B6.

Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5'-phosphate (P5P/pyridoxal phosphate/the active form of vitamin B6) ligand within the protein's binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.