Pulmonary arterial hypertension (PAH) can be caused by pathogenic variants in the gene bone morphogenetic protein receptor 2 (BMPR2). While BMPR2 protein expression levels are known to be reduced in the lung tissue of heritable PAH (HPAH) patients, a systematic study evaluating expression in more easily accessible blood samples and its clinical relevance is lacking. Thus, we analyzed the BMPR2 mRNA expression in idiopathic/HPAH patients and healthy controls in blood by quantitative polymerase chain reaction and protein expression by enzyme-linked immunosorbent assay. Clinical parameters included right heart catherization, echocardiography, six-minute walking test and laboratory tests. BMPR2 variant-carriers (n = 23) showed significantly lower BMPR2 mRNA expression in comparison to non-carriers (n = 56) and healthy controls (n = 30; p < 0.0001). No difference in BMPR2 protein expression was detected. Lower BMPR2 mRNA expression correlated significantly with greater systolic pulmonary artery pressure and pulmonary vascular resistance. Higher BMPR2 mRNA expression correlated with greater glomerular filtration rate, cardiac index and six-minute walking distance. We demonstrated the feasibility to assess BMPR2 expression in blood and, for the first time, that BMPR2 mRNA expression levels are significantly reduced in variant carriers and correlated with clinical parameters. Further studies may evaluate the usefulness of BMPR2 mRNA expression in blood as a new marker for disease severity.

Indiana University-Purdue University Indianapolis (IUPUI) / The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen which readily establishes itself in the lungs of people with cystic fibrosis (CF). Most CF patients have life-long P. aeruginosa infections. By modulating its own virulence and forming biofilms, P. aeruginosa is able to evade both host immune responses and antibiotic treatments. Previous studies have shown that the magnesium transporter MgtE plays a role in virulence modulation by inhibiting transcription of the type III secretion system, a mechanism by which bacteria inject toxins directly into the eukaryotic host cell. MgtE had already been identified as a magnesium transporter, and thus its role in regulating cytotoxicity was indicative of dual functions for this protein. This research focused on a structure-function analysis of MgtE, with the hypothesis that the magnesium transport and cytotoxicity functions could be exerted independently. Cytotoxicity assays were conducted using a co-culture model system of cystic fibrosis bronchial epithelial cells and a ∆mgtE strain of P. aeruginosa transformed with plasmids carrying wild type or mutated mgtE. Magnesium transport was assessed using the same mgtE plasmids in a Salmonella strain deficient in all magnesium transporters. Through analysis of a number of mgtE mutants, we found two constructs – a mutation in a putative magnesium binding site, and an N-terminal truncation – which demonstrated a separation of functions. We further demonstrated the uncoupling of functions by showing that different mgtE mutants vary widely in their ability to regulate cytotoxicity, whether or not they are able to transport magnesium. Overall, these results support the hypothesis of MgtE as a dual function protein and may lead to a better understanding of the mechanisms underlying P. aeruginosa virulence. By understanding virulence mechanisms, we may be able to develop treatments to reduce infections and pave the way to better health for people with cystic fibrosis.

Pseudomonas aeruginosa is a central nosocomial pathogen that can infect any tissue in the human body. A two-component system in P. aeruginosa that regulates many virulence factors is the AlgZ/R system. A previously unidentified regulator of algZ, that does not affect algR, has been identified via transposon mutagenesis, ‘PA2771’. The mechanism of regulation has not been previously studied, and novel evidence of PA2771 functioning as a diguanyalate cyclase was observed. When PA2771 is active, cyclic di-GMP levels are high, promoting the upregulation of the fimU operon and Type VI pili. In the PA2771 mutant, an upregulation in the expression of the flagellar genes and swarming phenotype was observed, and restored via complementation. PA2771's function in regulating algZ expression, is likely indirect and alters virulence gene regulation and phenotypic outputs in P. aeruginosa in the switch between twitching and swimming motility, and appears to be specific to PA2771.

Pseudomonas aeruginosa is a ubiquitous, opportunistic pathogen that causes acute and chronic infections. Infection is typically initiated via motile and virulent strains. After exposure to stressors, acute infections make both genotypic and phenotypic switches to a chronic, sessile strain. This is due to intricate regulatory networks directing gene expression in response to stressors. One network, GacA/GacS, has been established to control virulence factors. The stringent response of bacteria is mediated by alarmones produced primarily by RelA which responds to starvation. To study the effect of the stringent response on the virulence switch. A series of experiments were run in both PAO1, a virulent strain, and PDO300, an acute strain, and RelA deletion mutants of each transcriptional fusions of GacA/GacA system were integrated in the wild-types and mutants. Alginate, swimming, twitching, and biofilm formation assays were performed on all. The preliminary data suggests that the stringent response influences the GacA/GacS system.

The Type III Secretion (T3S) system is a system utilized by many pathogenic bacteria to inject proteins into host cells during an infection. Effector proteins enter the host cell by passing through the proteinaceous T3S translocon, which forms a pore on the host cell membrane. Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the T3S system, and very little is known about how the P. aeruginosa translocon forms. The proteins PopB and PopD are believed to assemble into the P. aeruginosa translocon. A pore-forming heterocomplex of PopB and PopD has been reconstituted in model membranes, however this heterocomplex has not been assessed in its relation to the translocon formed on the host cell. The interaction of this heterocomplex with other T3S system components was measured to determine if this complex acts similarly to the translocon. Initial assays that can be used to compare the molecular weight of the translocon isolated from eukaryotic cells after P. aeruginosa contact to the calculated molecular weight of the heterocomplex were developed as well. This study provides insight into how the PopB:PopD heterocomplex formed in model membranes relates to the translocon formed during a P. aeruginosa infection.

During the past months much has been published in periodicals, especially those of a pictorial nature, which discredits institutions established for the care of' the mentally ill throughout the nation. Many articles have been presented which show an intelligent approach, but the majority of these treatments are by those who are basing too much on a very narrow experience. For example, one news magazine recently printed a picture of a mental patient in institutional garb sitting on a bench in one of the poses most characteristic of a catatonic. The caption of the picture seemed to ignore this significant fact and instead to imply that the dejected pose was due to improper treatment. In this paper the author is neither defending the present mental institution, nor is he attempting to throw a "verbal brickbat" at the constructive work which is being done at institutions of this type. He is, rather, submitting an analysis of the prevalence and distribution of certain parasitic Protozoa in a large state hospital in the hope that the findings are of significance not merely in terms of institutional welfare but also in the more general terms of: (1) relationship between mental health and parasitic infections, and (2) regional differences in the incidence of such infections in California.

Colonization is prerequisite for infection and transmission of Streptococcus pneumoniae, or pneumococcus. Currently available pneumococcal conjugate and pneumococcal polysaccharide vaccines can provide protection against a limited number of capsular serotypes. Implementation of vaccines has decreased the frequency of invasive pneumococcal disease and their colonization rates, but only in a serotype-dependent manner. This has led to serotype replacement in pneumococcal ecology and increased invasive disease caused by non-vaccine serotypes. Development of conserved protein-based vaccine that can provide protection against all pneumococcal serotypes is needed. Numerous surface proteins are conserved in all serotypes, and some are known to be involved in the colonization process. Understanding how pneumococcal surface proteins interact with host cells and determining their roles in colonization will aid in vaccine development. In this dissertation, we characterized host cell receptors of pneumococcal surface proteins, and proteins involved in biofilm formation, and their effect in colonization. We utilized a novel protein expression vector, pOS1, which can express secreted proteins with no LPS, IPTG induction, or cell lysis requirement. These expressed recombinant proteins were used for further investigation. We identified that human Annexin A2 (ANXA2) interacts with pneumococcal surface adhesion A (PsaA) protein. ANXA2 transduced cells showed significant increase in binding with pneumococcus compared to non-transduced cells. We conducted proteomic profiling of planktonic and biofilm membrane proteins and identified that two lipoproteins (AmiA, SP_0148) were overexpressed during biofilm formation. Isogenic mutants lacking these individual proteins showed decreased in biofilm formation compared to their parental strain. Deletion of SP_0148 led to decreased adhesion of pneumococcus to human nasopharyngeal epithelial cells (Detroit 562). These results increased our understanding of pneumococcal surface proteins involved in biofilm-formation and colonization as well as identifying new host receptors ligands for these adhesins.

Avian pathogenic Escherichia coli and Clostridium perfringens cause economic and welfare concerns to the broiler industry. The recent shift to no antibiotics ever (NAE) production has increased disease incidence. The objectives of this study were to determine the influence of season, age of flock, and sample type on E. coli prevalence and virulence and to identify C. perfringens prevalence and toxinotypes in NAE farms. Results indicated high prevalence of virulent E. coli; prevalence of virulent E. coli decreased from Spring to Summer. Virulent E. coli showed high resistance to antimicrobials. Serogroups O8 and O78 were most prevalent in virulent E. coli. C. perfringens prevalence was very low and all recovered isolates were toxinotype A with variation in netB, cpb2, and tpeL presence. In conclusion, NAE farms should have measures to control E. coli infections, especially in spring season. Further studies are required to confirm the lower prevalence of C. perfringens.

Bacillus anthracis is a soil dwelling microbe with pronounced pathogenic potential. Historically, anthrax has infected livestock and man. In the modern-age, anthrax is a bioterrorism concern with major incidents every decade. While the threat of large scale attacks is currently viewed as unlikely, the threat is consistent and constant. Current methods to defend against such an attack focus on antibiotics and containment of public panic. Antibiotic resistance, while not currently an issue for anthrax, could easily become so with genetically engineered weaponized strains created by rogue states or independent actors. This project evolved from collaborations between the Grose lab and the Robison lab, both housed in the Microbiology and Molecular Biology Department at Brigham Young University in Provo, Utah. Two undergraduates in the Grose lab isolated 23 genetically distinct phage that infect the non-pathogenic Bacillus anthracis Sterne strain. Results from spot testing on a diverse library of 11 fully virulent strains that represent the extant genetic diversity of pathogenic B. anthracis in BYU’s BSL-3 facility give credence to the idea that phage could be useful in containing this pathogen. Phage were isolated from environmental samples using enrichment culture, high titer lysates of isolated phage were created, and differential assays were performed. Experiments to show phage differences included electron microscopy, restriction digests, and spot testing using different isolates of B. anthracis. These data identified several novel phage that could infect a wide variety of virulent B. anthracis isolates. Preliminary results also showed most of these phage to be different both morphologically and genetically.We propose that phage therapy deserves further research, public awareness, and increased understanding for governmental regulatory awareness.

There is an emerging threat of Neisseria gonorrhoeae strains that are resistant to all antibiotics. Because of this, the purpose of this research is to isolate, analyze, and partially characterize a new inhibitor(s) of N. gonorrhoeae. Since there is an unknown molecule secreted by Candida albicans that inhibits N. gonorrhoeae, this molecule can be partially characterized using 1H NMR Spectroscopy to assist in the development of a new antibiotic compound. It was hypothesized that quorum-sensing molecules, trans, trans- farnesol, tyrosol, phenylethyl alcohol, and tryptophol, could be possible candidates for the inhibitor. Because of this, 1H NMR spectra for these quorum-sensing molecules were obtained to serve as controls. Column chromatography and fractionation was used to isolate the inhibitor in large scale from C. albicans grown in salts-based media. Attempts to isolate the inhibitor in large scale, however, was unsuccessful since no inhibition of N. gonorrhoeae was observed. Because of this, analysis of growth media was conducted to test the media effect on producing the inhibitor. C. albicans was grown in liquid chocolate, liquid white chocolate, salts-based, and YPD media in aerobic and candle jar environments. Analysis of growth media in different environments suggests that liquid chocolate and salts-based media retain the inhibitory activity. 1H NMR spectra were obtained for the isolated molecule in liquid chocolate and salts-based media in both aerobic and candle jar environments. Analysis of this 1H NMR suggested that the inhibitor could be isolated from either the aerobic or candle jar environment for both liquid chocolate and salt-based media because a clear peak between 3.5 and 4.0 ppm was observed in all spectra. Comparison of 1H NMR spectra from quorum-sensing molecules with spectra from the isolated molecule suggests that the inhibitor is not a quorum-sensing molecule. The peaks represented by the inhibitor cannot be fully characterized and thus, either correspond to a single molecule or a complex molecular structure. It can be concluded that the inhibitor secreted by C. albicans to inhibit N. gonorrhoeae is a new unknown compound.