Bacterial genome engineering and synthetic biology: combating pathogens.

BACKGROUND: The emergence and prevalence of multidrug resistant (MDR) pathogenic bacteria poses a serious threat to human and animal health globally. Nosocomial infections and common ailments such as pneumonia, wound, urinary tract, and bloodstream infections are becoming more challenging to treat due to the rapid spread of MDR pathogenic bacteria. According to recent reports by the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC), there is an unprecedented increase in the occurrence of MDR infections worldwide. The rise in these infections has generated an economic strain worldwide, prompting the WHO to endorse a global action plan to improve awareness and understanding of antimicrobial resistance. This health crisis necessitates an immediate action to target the underlying mechanisms of drug resistance in bacteria. RESEARCH: The advent of new bacterial genome engineering and synthetic biology (SB) tools is providing promising diagnostic and treatment plans to monitor and treat widespread recalcitrant bacterial infections. Key advances in genetic engineering approaches can successfully aid in targeting and editing pathogenic bacterial genomes for understanding and mitigating drug resistance mechanisms. In this review, we discuss the application of specific genome engineering and SB methods such as recombineering, clustered regularly interspaced short palindromic repeats (CRISPR), and bacterial cell-cell signaling mechanisms for pathogen targeting. The utility of these tools in developing antibacterial strategies such as novel antibiotic production, phage therapy, diagnostics and vaccine production to name a few, are also highlighted. CONCLUSIONS: The prevalent use of antibiotics and the spread of MDR bacteria raise the prospect of a post-antibiotic era, which underscores the need for developing novel therapeutics to target MDR pathogens. The development of enabling SB technologies offers promising solutions to deliver safe and effective antibacterial therapies.

Proteomic Analysis of Non-human Primate Peripheral Blood Mononuclear Cells During Burkholderia mallei Infection Reveals a Role of Ezrin in Glanders Pathogenesis.

Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1ß (IL-1ß). On the contrary, the expression of IL-1ß receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1ß production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model.

Mitigating the Impact of Antibacterial Drug Resistance through Host-Directed Therapies: Current Progress, Outlook, and Challenges.

Increasing incidences of multidrug resistance in pathogenic bacteria threaten our ability to treat and manage bacterial infection. The development and FDA approval of novel antibiotics have slowed over the past decade; therefore, the adoption and improvement of alternative therapeutic strategies are critical for addressing the threat posed by multidrug-resistant bacteria. Host-directed therapies utilize small-molecule drugs and proteins to alter the host response to pathogen infection. Here, we highlight strategies for modulating the host inflammatory response to enhance bacterial clearance, small-molecule potentiation of innate immunity, and targeting of host factors that are exploited by pathogen virulence factors. Application of state-of-the-art "omic" technologies, including proteomics, transcriptomics, and image-omics (image-based high-throughput phenotypic screening), combined with powerful bioinformatics tools will enable the modeling of key signaling pathways in the host-pathogen interplay and aid in the identification of host proteins for therapeutic targeting and the discovery of host-directed small molecules that will regulate bacterial infection. We conclude with an outlook on research needed to overcome the challenges associated with transitioning host-directed therapies into a clinical setting.

Teen Crashes Declined After Massachusetts Raised Penalties For Graduated Licensing Law Restricting Night Driving.

In 2007, as part of the Massachusetts graduated driver-licensing program designed to allow junior operators (ages 16½-17 years) to gain experience before receiving full licensure, stringent penalties were introduced for violating a law prohibiting unsupervised driving at night; driver education, including drowsy driving education, became mandatory; and other new restrictions and penalties began. We evaluated the impact of these changes on police-reported vehicle crash records for one year before and five years after the law's implementation in drivers ages 16-17, inclusive, and two comparison groups. We found that crash rates for the youngest drivers fell 18.6 percent, from 16.24 to 13.22 per 100 licensed drivers. For drivers ages 18-19 the rates fell by 6.7 percent (from 9.59 to 8.95 per 100 drivers), and for those ages 20 and older, the rate remained relatively constant. The incidence rate ratio for drivers ages 16-17 relative to those ages 20 and older decreased 19.1 percent for all crashes, 39.8 percent for crashes causing a fatal or incapacitating injury, and 28.8 percent for night crashes. Other states should consider implementing strict penalties for violating graduated driver-licensing laws, including restrictions on unsupervised night driving, to reduce the risk of sleep-related crashes in young people.