Violeta Chavez

Oxidative Stress Enzymes Are Required for DAF-16-Mediated Immunity Due to Generation of Reactive Oxygen Species by Caenorhabditis elegans

Caenorhabditis elegans has recently been developed as a model for microbial pathogenesis, yet little is known about its immunological defenses. Previous work implicated insulin signaling in mediating pathogen resistance in a manner dependent on the transcriptional regulator DAF-16, but the mechanism has not been elucidated. We present evidence that C. elegans, like mammalian phagocytes, produces reactive oxygen species (ROS) in response to pathogens. Signs of oxidative stress occur in the intestine—the site of the host–pathogen interface—suggesting that ROS release is localized to this tissue. Evidence includes the accumulation of lipofuscin, a pigment resulting from oxidative damage, at this site. In addition, SOD-3, a superoxide dismutase regulated by DAF-16, is induced in intestinal tissue after exposure to pathogenic bacteria. Moreover, we show that the oxidative stress response genes sod-3 and ctl-2 are required for DAF-16-mediated resistance to Enterococcus faecalis using a C. elegans killing assay. We propose a model whereby C. elegans responds to pathogens by producing ROS in the intestine while simultaneously inducing a DAF-16-dependent oxidative stress response to protect adjacent tissues. Because insulin-signaling mutants overproduce oxidative stress response enzymes, the model provides an explanation for their increased resistance to pathogens.

The Molecular Alarmone (p)ppGpp Mediates Stress Responses, Vancomycin Tolerance, and Virulence in Enterococcus faecalis

The stringent response is a global bacterial response to stress that is mediated by accumulation of the alarmone (p)ppGpp. In this study, treatment with mupirocin was shown to induce high levels of (p)ppGpp production in Enterococcus faecalis, indicating that this nosocomial pathogen can mount a classic stringent response. In addition, (p)ppGpp was found to accumulate in cells subjected to heat shock, alkaline shock, and inhibitory concentrations of vancomycin. Sequence analysis of the E. faecalis genome indicated that (p)ppGpp synthesis is catalyzed by the bifunctional synthetase/hydrolase RelA and the RelQ small synthase. The (p)ppGpp profiles of DeltarelA, DeltarelQ, and DeltarelAQ strains revealed that RelA is the major enzyme responsible for the accumulation of (p)ppGpp during antibiotic or physical stresses, while RelQ appears to be responsible for maintaining basal levels of alarmone during homeostatic growth. Compared to its parent, the DeltarelA strain was more susceptible to several stress conditions, whereas complete elimination of (p)ppGpp in a DeltarelAQ double mutant restored many of the stress-sensitive phenotypes of DeltarelA. Interestingly, growth curves and time-kill studies indicated that tolerance to vancomycin is enhanced in the DeltarelA strain but diminished in the DeltarelQ and DeltarelAQ strains. Finally, virulence of the DeltarelAQ strain but not of the DeltarelA or DeltarelQ strain was significantly attenuated in the Caenorhabditis elegans model. Taken together, these results indicate that (p)ppGpp pools modulate environmental stress responses, vancomycin tolerance, and virulence in this important nosocomial pathogen.

Ce-Duox1/BLI-3 Generates Reactive Oxygen Species as a Protective Innate Immune Mechanism in Caenorhabditis elegans

ABSTRACT Caenorhabditis elegans was recently developed as a model system to study both pathogen virulence mechanisms and host defense responses. We previously demonstrated that C. elegans produces reactive oxygen species (ROS) in response to exposure to the important gram-positive nosocomial pathogen Enterococcus faecalis. We also presented evidence of oxidative stress and upregulation of stress responses after exposure to the pathogen. As in mammalian systems, this new work shows that production of ROS for innate immune functions occurs via an NADPH oxidase. Specifically, reducing expression of a dual oxidase, Ce-Duox1/BLI-3, causes a decrease in ROS production in response to E. faecalis. We also present evidence that reduction of expression of Ce-Duox1/BLI-3 increases susceptibility to this pathogen, specifically when expression is reduced in the intestine and the hypodermis. Ce-Duox1/BLI-3 was previously characterized as having a role in cuticle cross-linking. Two C. elegans mutants with point mutations in the peroxidase domain that exhibit severe cuticle defects were discovered to be unaffected in ROS production or pathogen susceptibility. These results demonstrate an important biological role for the peroxidase domain in cuticle cross-linking that is unrelated to ROS production. To further demonstrate the protective effects of the pathogen-induced ROS production, we show that antioxidants that scavenge ROS increase the sensitivity of the nematode to the infection, in stark contrast to their longevity-promoting effects under nonpathogenic conditions. In conclusion, we postulate that the generation of ROS by NADPH oxidases in the barrier epithelium is an ancient, highly conserved innate immune defense mechanism.

Localization of the Dual Oxidase BLI-3 and Characterization of Its NADPH Oxidase Domain during Infection of Caenorhabditis elegans.

Dual oxidases (DUOX) are enzymes that contain an NADPH oxidase domain that produces hydrogen peroxide (H2O2) and a peroxidase domain that can utilize H2O2 to carry out a variety of reactions. The model organism Caenorhabditis elegans produces the DUOX, BLI-3, which has roles in both cuticle development and in protection against infection. In previous work, we demonstrated that while certain peroxidases were protective against the human bacterial pathogen Enterococcus faecalis, the peroxidase domain of BLI-3 was not, leading to the postulate that the NADPH oxidase domain is the basis for BLI-3’s protective effects. In this work, we show that a strain carrying a mutation in the NADPH oxidase domain of BLI-3, bli-3(im10), is more susceptible to E. faecalis and the human fungal pathogen Candida albicans. Additionally, less H2O2 is produced in response to pathogen using both an established Amplex Red assay and a strain of C. albicans, WT-OXYellow, which acts as a biosensor of reactive oxygen species (ROS). Finally, a C. elegans line containing a BLI-3::mCherry transgene was generated. Previous work suggested that BLI-3 is produced in the hypodermis and the intestine. Expression of the transgene was observed in both these tissues, and additionally in the pharynx. The amount and pattern of localization of BLI-3 did not change in response to pathogen exposure.

Dirofilariasis Presenting as an Infiltrative Mass in the Right Buccal Space

Dirofilariasis is caused by filarial nematodes (roundworms) of the genus Dirofilaria. Dirofilariasis of the oral mucosa is very rare. Herein, we report a case of a 79-year-old man who had a slowly growing infiltrative mass in the right buccal space. Histopathologic examination showed an inflammatory infiltrate with eosinophilia, histiocytes, and small organisms (0.2-0.3 mm). Digital images were sent to the Centers for Disease Control and Prevention, which identified the parasite as a nematode in the genus Dirofilaria. It appeared to be dead and degenerating, but external, fine longitudinal cuticular ridges and the presence of tall muscle cells were diagnostic. Thus, Dirofilaria, despite its rarity, should be considered in the differential diagnosis of tumor-like lesions in the buccal mucosa.

Carbapenem‑resistant Lactobacillus intra‑abdominal infection in a renal transplant recipient with a history of probiotic consumption

BackgroundLactobacillus sp. is a low virulence bacterium, which rarely causes infection in immunocompetent individuals and usually is considered a contaminant. Normally this organism is susceptible to β-lactam antibiotics, yet resistant strains have been reported.Case presentation and discussionHere, we report a case of a 60-year-old renal transplant recipient who developed an intra-abdominal abscess which grew a carbapenem-resistant Lactobacillus casei. This is significant since it is the first report of a clinical isolate of Lactobacillus sp. that demonstrated both microbiological and clinical resistance to carbapenem use. Moreover, the probiotic supplement that the patient had taken also grew a similar organism raising the concern of probiotic associated infection in immunocompromised individual.

Overview of Bacteria

This chapter will deal with all common Gram-positive and Gram-negative bacteria, as well as Enterobacteriaceae, anaerobes, and mycobacteria and their role in infective patients. Drugs which are used in treating such infections will also be addressed.

Acute appendicitis caused by Schistosoma japonicum

74-year-old woman presented with right lower uadrant abdominal pain and diarrhea for 3 days. he was otherwise healthy and had recently reloated from Philippines to the United States. Her hite blood cell count was normal, and testing or human immunodeficiency virus was negative. omputed tomography showed acute appendiciis (Fig. 1a, arrow), cecal wall thickening and at stranding. During laparoscopic appendectomy, he cecum was noted to be adherent to the latral abdominal wall. The appendix was short and dherent to the surrounding fat without signs of erforation or pus. Histopathological examination f the appendix showed oval foreign bodies (Fig. 1b

Overview of Molecular Diagnostics Principles

This chapter will provide the fundamental knowledge of molecular diagnostics needed for a trainee during board preparation and the general practicing pathologist and will briefly discuss the basic principles of molecular biology and outline the basic techniques used in molecular diagnostics. This chapter will also touch on the available molecular diagnostic instruments and the design of a clinical molecular laboratory.

Specific Clinical Infections

This chapter will discuss various microbiological aspects of urinary tract infection, gastrointestinal tract infections, respiratory tract infections, endocarditis, infection of the nervous system, joint infection, hepatitis, bacteremia, and septicemias.