Acinetobacter baumannii is an important human pathogen due to its multi-drug resistance. In this study, the genome of an ST10 outbreak A. baumannii isolate LAC-4 was completely sequenced to better understand its epidemiology, antibiotic resistance genetic determinants and potential virulence factors. Compared with 20 other complete genomes of A. baumannii, LAC-4 genome harbors at least 12 copies of five distinct insertion sequences. It contains 12 and 14 copies of two novel IS elements, ISAba25 and ISAba26, respectively. Additionally, three novel composite transposons were identified: Tn6250, Tn6251 and Tn6252, two of which contain resistance genes. The antibiotic resistance genetic determinants on the LAC-4 genome correlate well with observed antimicrobial susceptibility patterns. Moreover, twelve genomic islands (GI) were identified in LAC-4 genome. Among them, the 33.4-kb GI12 contains a large number of genes which constitute the K (capsule) locus. LAC-4 harbors several unique putative virulence factor loci. Furthermore, LAC-4 and all 19 other outbreak isolates were found to harbor a heme oxygenase gene (hemO)-containing gene cluster. The sequencing of the first complete genome of an ST10 A. baumannii clinical strain should accelerate our understanding of the epidemiology, mechanisms of resistance and virulence of A. baumannii.

Trichomonas vaginalis (Tv) is an extracellular protozoan parasite that causes the most common non-viral sexually transmitted infection: trichomoniasis. While acute symptoms in women may include vaginitis, infections are often asymptomatic, but can persist and are associated with medical complications including increased HIV susceptibility, infertility, pre-term labor, and higher incidence of cervical cancer. Heightened inflammation resulting from Tv infection could account for these complications. Effective cellular immune responses to Tv have not been characterized, and re-infection is common, suggesting a dysfunctional adaptive immune response. Using primary human leukocyte components, we have established an in vitro co-culture system to assess the interaction between Tv and the cells of the human immune system. We determined that in vitro, Tv is able to lyse T-cells and B-cells, showing a preference for B-cells. We also found that Tv lysis of lymphocytes was mediated by contact-dependent and soluble factors. Tv lysis of monocytes is far less efficient, and almost entirely contact-dependent. Interestingly, a common symbiont of Tv, Mycoplasma hominis, did not affect cytolytic activity of the parasite, but had a major impact on cytokine responses. M. hominis enabled more diverse inflammatory cytokine secretion in response to Tv and, of the cytokines tested, Tv strains cleared of M. hominis induced only IL-8 secretion from monocytes. The quality of the adaptive immune response to Tv is therefore likely influenced by Tv symbionts, commensals, and concomitant infections, and may be further complicated by direct parasite lysis of effector immune cells.

The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.

Telomeres are structures at the ends of chromosomes that shorten during cell division and eventually signal an irreversible state of growth arrest known as cellular senescence. To delay this cellular aging, human T cells, which are critical in the immune control over infections and cancer, activate the enzyme telomerase, which binds and extends the telomeres. Several different extracts from the Astragalus membranaceus root have been documented to activate telomerase activity in human T cells. The objective of this research was to compare two extracts from Astragalus membranaceus, TA-65 and HTA, for their effects on both telomerase and proliferative activity of human CD4 and CD8 T cells. Our results demonstrate that, TA-65 increased telomerase activity significantly (1.3 to 3.3-fold relative to controls) in T cell cultures from six donors tested, whereas HTA only increased telomerase levels in two out of six donors. We also demonstrate that TA-65 activates telomerase by a MAPK- specific pathway. Finally, we determine that during a three-day culture period, only the T cells treated with the TA-65 extract showed a statistically significant increase in proliferative activity. Our results underscore the importance of comparing multiple telomerase activators within the same experiment, and of including functional assays in addition to measuring telomerase activity.