Scrub typhus is a potentially fatal infection that threatens one billion persons in the Asia-Pacific region and is caused by the obligate intracellular bacterium, Orientia tsutsugamushi. How this organism facilitates its intracellular survival and pathogenesis is poorly understood. Intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) into the host cell to modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank libraries as well as Type 1 and Type 4 secretion systems (T1SS and T4SS), which are expressed during infection. In silico analyses of the Anks’ C-termini revealed that they possess characteristics of T1SS secretion signals. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks. In addition to infecting endothelial cells, O. tsutsugamushi infects professional phagocytes. To better understand why these innate immune cells are unable to eliminate O. tsutsugamushi, we addressed the activity of host NF-κB proinflammatory transcription factor. Screening of O. tsutsugamushi infected cells at an MOI of 1 revealed inhibition of NF-κB nuclear accumulation as early as 8 hours in HeLa and bone-marrow derived macrophage cells. When stimulating infected cells with TNF-α, IκBα degradation still occurs, however NF-κB dependent gene transcription remains downregulated. Immunofluorescence microscopic analysis of TNF-α treated cells ectopically expressing all O. tsutsugamushi Anks revealed that two nuclear trafficking Anks, Ank1 and Ank6, result in a significant decrease in NF-κB nuclear accumulation. Additionally, these Anks also significantly inhibited NF-κB dependent gene transcription. Co-immunoprecipitation experiments revealed that both Anks interact with importin-β1, exportin-1, and the p65 NF-κB subunit. Treating cells with importazole significantly reduces the nuclear accumulation of Ank1 and Ank6. Finally, treating infected cells or cells ectopically expressing Ank1 or Ank6 with leptomycin B resulted in restoration of NF-κB nuclear accumulation. With these data, we propose that O. tsutsugamushi secretes Ank1 and Ank6 to initially interact with importin-β1, which permits their nuclear entry where they then interact with NF-κB and subsequently exportin-1 to prevent NF-κB nuclear accumulation.

Klebsiella pneumoniae is an opportunistic pathogen responsible for lobar pneumoniae, liver abscess, and septicemia. Clinical isolates are found to be extended spectrum beta lactamase positive with differential expression of the two classical porins, OmpK35 and OmpK36. Porin loss is associated with increased minimum inhibitory concentrations of beta lactam, cephalosporin, and carbapenem antibiotics that target the peptidoglycan. However, little is known about how porin loss affects other aspects of the cell envelope. The focus of this study was to characterize clinical isolates exhibiting differential porin expression and determine if the cumulative changes altered the resistance to phagocytosis by macrophages. The results support the hypothesis that porin loss significantly impacts the overall cell envelope composition, which in turn alters interactions with macrophages.

This study investigated the antimicrobial efficacy of bioactive plant compounds along with high pressure processing (HPP) against pathogens Bacillus cereus and Cronobacter sakazakii in infant formula and infant rice cereal. The influence of these applications on antimicrobial activity, shelf-life and sensory attributes of infant foods were examined. Trans-cinnamaldehyde (TC), (-)-Epigallocatechin gallate (EGCG) and [10]-Gingerol (GI) were incorporated (0.05%) in infant rice cereal reconstituted with infant formula. The cereal was inoculated with either B. cereus (ATCC 14579) or B. cereus spores (107-108 log CFU g-1). All the samples were stored at 7, 23 or 37°C for 0, 4, 8 and 24 h. TC showed the highest antimicrobial activity by inhibiting the B. cereus and its spores up to 2.72 and 3.8 log CFU g-1, respectively. HPP (600 MPa for 5 m), and TC (0.05-0.1%) along with Chitosan (CH) (1%), were applied to reconstituted powder infant formula which was inoculated with either 3 strains of C. sakazakii (ATCC 29544, ATCC 12868, and ATCC BAA 894) or 5 strains of B. cereus spore (ATCC 14579, ATCC 33018, ATCC 12826, ATCC 4342, and Difco Spores) cocktail (107-108 log CFU ml-1). All the samples were stored at 7, 23 or 45°C for 5-8 weeks. HPP and TC (0.1%) combination exhibited the highest inhibition (P < 0.05) by reducing the B. cereus spores 2.97 log CFU ml-1 after 7 d. C. sakazakii was fully inactivated by HPP, TC (0.05%) and C (1%) combination following 8 weeks of storage at 7 and 23°C and 2 weeks storage at 45°C. The combination of HPP and bioactive compounds exhibited additive antimicrobial effect. Gradual decrease (P < 0.05) in pH was observed in rice cereal and non-HPP formula samples due to the microbial growth and metabolic activity. Significant differences (P < 0.05) were found in color, aroma and general appearance of EGCG and GI applied cereal samples, while TC only did exhibit a cinnamon taste. In summary, the antimicrobial findings suggest that TC, EGCG, GI and CH could be incorporated in infant foods along with HPP as natural and safe alternatives to synthetic preservatives and thermal applications.

Chlamydia trachomatis serovars D-K are the leading cause of bacterially-acquired sexually transmitted infections in the United States. As an obligate intracellular pathogen, C. trachomatis infects columnar epithelial cells of the genital mucosae and can cause deleterious sequelae such as pelvic inflammatory disease, infertility, and ectopic pregnancy. Several chlamydial antigens reach the host cell cytosol prior to the natural release of chlamydiae at the end of the developmental cycle. While some of these extra-inclusion antigens traffic to the host cell surface, others remain intracellular where they are proposed to influence vital host cell functions and antigen trafficking and presentation. The research herein examines the escape and trafficking of the immunodominant chlamydial antigens MOMP, LPS, and cHsp60 within C. trachomatis serovar E-infected polarized human endometrial epithelial cells. Studies using high-resolution transmission electron microscopy (TEM) and immuno-TEM report the novel escape mechanism of chlamydial antigens via vesicles everted/pinched off from the inclusion membrane, an occurrence observed both in the presence and absence of the antibiotic azithromycin. These extra-inclusion vesicles were differentiated from Golgi vesicles and were shown to deliver chlamydial heat shock protein 60 (cHsp60)-homologs 2 and 3, but not homolog 1, to the infected cell surface. Examination of the iron-responsiveness of the three cHsp60 homologs by immuno-TEM revealed a significant increase in cHsp60-2 following iron deprivation. Further investigation of the trafficking of chlamydial MOMP and LPS antigens enveloped within the protective everted inclusion membrane vesicles within host cells involved density gradient centrifugation for the separation of epithelial secretory pathway components followed by SDS-PAGE and Western blot to determine whether the chlamydial antigen-containing vesicles could fuse with and deliver the antigens to host cell organelles. Coupled with immuno-TEM, these data confirmed the presence of major chlamydial antigens within the endoplasmic reticulum of infected host cells. Additionally, chlamydial lipopolysaccharide (LPS) was co-localized with CD1d, a lipid antigen-presenting molecule. Collectively, these studies (i) establish a novel escape mechanism for chlamydial antigens, (ii) identify cHsp60-2 as a marker of iron stress response in C. trachomatis, and (iii) define for the first time the host cell ER as a destination for selected chlamydial antigens during infection.

More so than ever, Multiple Drug Resistant (MDR) bacteria are on the rise due to overuse of antibiotics along with natural selection for adaptations that enhance drug-resistant properties. One particular bacterial family, Enterobacteriaceae, has been problematic, exhibiting several bacterial members that have developed a precipitous resistance to modern antibiotics and are also primary causative agents of nosocomial, or hospital acquired, infections. Citrobacter freundii (CF) and Enterobacter cloacae (ECL) are two species of the Enterobacteriaceae family causing significant medical concern due to their role in producing numerous opportunistic infections such as bacteremia, lower respiratory tract infections, urinary tract infections, and endocarditis. Adding to the difficulty of this situation is the ability of bacteria to produce biofilms. These biofilms are communities of bacteria that exhibit increased resistance to antibiotic treatment and eradication. Previous work in the laboratory of Dr. Fox at ETSU has identified an uncharacterized product secreted by Klebsiella pneumoniae (KP), another member of the Enterobacteriaceae family, which appears to have inhibitory effects toward CF and ECL. The current study was designed to characterize the effects this secreted product has on CF and ECL biofilms. Through a high throughput microtiter plate assay, the effects of this secreted product were examined on CF and ECL phases of biofilm attachment and maturation. Based on our findings, we have concluded that this secreted product can be categorized as a possible bacteriostatic agent against biofilm cell density, biofilm mass, and cell viability for both biofilm phases of attachment and maturation.

Staphylococcus pseudintermedius affecting dogs is analogous to S. aureus on humans, acting as both normal flora and opportunistic pathogen. Methicillin resistance in S. pseudintermedius is recent, with the first documented occurrence of an isolate bearing the methicillin resistance gene, mecA, in 1999. This gene encodes penicillin binding protein 2a, which renders all beta-lactam drugs ineffective and functions as a “gateway” antibiotic resistance determinant. In the presence of ineffective antibiotics, opportunities for mutational events and acquisition of mobile genetic elements increase as microbial densities increase, often leading to multi-drug resistance. Methicillin-resistant S. pseudintermedius (MRSP) infections have become increasingly common. For example, approximately 30% of the S. pseudintermedius isolates tested by the University of Tennessee College of Veterinary Medicine Clinical Bacteriology Laboratory are resistant to methicillin. An increasing number of MRSP isolates are also resistant to most clinically useful antibiotics available to veterinarians except for chloramphenicol, and resistance to this antibiotic is common among European MRSP isolates. Chloramphenicol resistance has begun to appear in the US and if this trend continues there may soon be few viable antibiotic treatment options. Compared with the arrival of methicillin-resistant S. aureus in the 1960s, the opportunity currently exists to apply advanced molecular methods early in this recognized emergence of MRSP. To that end I have pursued projects utilizing multilocus sequence typing, pulsed-field electrophoresis, and SCCmec characterization of both susceptible and resistant S. pseudintermedius. The initial result was the detection of a clonal population of MRSP in the southeastern United States. Further characterization of this and other clonal lineages using genomic sequencing and real-time RT-PCR expression analysis of antibiotic resistance and quorum sensing genes revealed a marked difference in the regulation of antibiotic resistance between regional clones. These discoveries have interesting epidemiological implications and provide a foundation for the development of novel therapeutics to circumvent the expanding antibiotic resistance repertoire of MRSP. Potential targets identified by this work include membrane-bound beta-lactamase receptors responsible for the regulation of mecA, non-cognate auto-inducing peptides, and synthetic antisense oligonucleotides.

The invasive pathways, InlA- and InB-mediated uptake and M cell transcytosis, that Listeria monocytogenes uses to invade the intestine have mainly been studied using infection models that do not truly replicate what occurs during a natural infection. Recently, our lab has developed an oral infection model that is more physiolocally relevant to what occurs during food borne listeriosis. We have sought to evaluate the relative roles of the previously defined invasive pathways, in our oral model of infection. We have done this by utilizing an InlAmCG Lm strain that is able to bind murine E-cadherin, knockout Lm strains, ΔinlA Lm, and ΔinlAΔinlB Lm. We also took advantage of a knockout mice strain CD137-/-that has M cells that are deficient in M cell transcytosis. We were able to show that these invasive pathways are relevant in our oral infection model, that M cell transcytosis is a compensatory pathway for InlA-mediated uptake, and that there might be another mechanism that L. monocytogenes uses to invade the intestines. To confirm this, it is necessary though that the M cell transcytosis deficiency be confirmed in the CD137-/- mice.

Planktothricoides raciborskii and Roseofilum reptotaenium are physiologically similar, yet ecologically distinct organisms found in a hot spring outflow and coral black band disease (BBD), respectively. The aim of this study was to elucidate the relationship between R. reptotaenium and sulfide in BBD, to compare microcystin (MC) production in response to environmental factors, and to determine chemotactic responses to MC and sulfide by the two organisms. Results showed that the pathogenicity of R. reptotaenium in BBD is dependent on sulfate-reducing bacteria as secondary pathogens. Roseofilum reptotaenium produced significantly more MC than P. raciborskii, as measured using ELISA. Roseofilum reptotaenium was negatively chemotactic to sulfide, determined using horizontal and vertical gradients in agar, while P. raciborskii was not affected. Neither cyanobacterium was chemotactic to MC in the agar assays. The ecophysiology of P. raciborskii and R. reptotaenium in relation to MC production and response to sulfide reflected their pathogenic versus non-pathogenic status.

Chlamydia is the most common bacterial sexually transmitted infection in the United States and worldwide. It often goes unnoticed due to lack of symptoms and left untreated it can ascend the female genital tract to cause sequelae like pelvic inflammatory disease and irreversible tubal infertility. In reproductive-aged women, female sex hormones estrogen (E2) and progesterone (P4) concentrations fluctuate during the menstrual cycle and are influenced by hormonal contraceptives and hormone replacement therapy. E2 and P4 influence genital Chlamydia infection in women and mice, but these multifactorial interactions are not entirely mapped out. The complex interplay of E2 and P4 with Chlamydia and the host response demand further study to determine the effect of hormonal environment and host susceptibility to Chlamydia. E2 primarily signals through estrogen receptors (ER) ERα and ERβ. We used ERα or ERβ knockout (KO) mice to study the role of E2 and ERs in chlamydial progression and examined the host immune response at day 9 post-infection, when we expected the immune response to be the most robust. ERαKO, but not ERβKO mice had significant differences in the progression of Chlamydia and the host immune response. Future studies should test the immune response at additional timepoints, and a model should be utilized wherein ERα and ERβ are simultaneously silenced by chemical knockdown of ERβ in ERα knockout mice using ER agonist ICI 182, 680. 3 Mice are widely used in Chlamydia research, but due to its short estrus cycle, infection cannot be established naturally before infected cells are shed. To overcome this, mice are pretreated with depot medroxyprogesterone acetate (DMPA), an exogenous progesterone that halts the estrus cycle. However, a mouse model not reliant on DMPA pretreatment is needed because 1.) DMPA can affect the immune response and 2.) the hormonal environment in women is not static. Our model uses mice that are ovariectomized to stop the production of endogenous E2 and P4, then treated with physiologically relevant levels of E2 and P4 via implantation of a hormone-filled capsule. We observed that E2 protected mice from Chlamydia, making our model a good alternative for in vivo Chlamydia studies.

Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we focused on the inhibitory properties of Farnesol for growth and motility of A. faecalis, as well as, the molecule’s ability to prevent Alcaligenes from creating biofilms and/or degrading them once they have already been established. Our experiments show evidence that Farnesol is able to inhibit both the growth and motility of A. faecalis, and determination of the specific concentrations of Farnesol needed to see the largest effects on A. faecalis biofilms. Our hope is that in future studies, we will be able to add varying concentrations of the Farnesol to known and widely used antibiotics in order to increase the effectiveness of antibiotics against bacterial strains, both in the Alcaligenes genus and in other genus, that have previously been considered “antibiotic resistant”.