Sexually transmitted infections and the vaginal microbiome after vaginoplasty.

PURPOSE OF REVIEW: Transgender and gender-diverse individuals (TGD) are at risk for sexually transmitted infections. Gender affirmation surgery is a cornerstone of care for many TGD individuals. For genital gender affirmation, the surgical creation of a vagina may be performed through a number of techniques. Those who have undergone vaginoplasty have unique anatomical and biopsychosocial considerations, which we discuss. RECENT FINDINGS: While sexually-transmitted infections including HPV, HSV, HIV, gonorrhea, and chlamydia, have been described in TGD individuals after vaginoplasty, the reports are very rare, and the provider should maintain an index of suspicion and maintain a broad differential for symptoms including neovaginal discharge. We discuss the association of the neovaginal microbiota composition with bacterial vaginosis, and how its modulation could potentially reduce bacterial vaginosis and sexually transmitted infection risk. SUMMARY: We examine the literature regarding sexually-transmitted infections following vaginoplasty, and the neovaginal microbiome and its similarities and differences relative to the natal vaginal microbiome.

Urinalysis is predictive for absence of urinary tract infection in men with and without catheters.

OBJECTIVES: To determine accuracy of negative urinalysis (UA) for predicting negative urine culture and the absence of urinary tract infection (UTI), and optimal urine culture growth cutoff for UTI diagnosis in men with and without urinary catheters. SUBJECTS AND METHODS: UAs with urine cultures within 1 week from adult men were identified and evaluated. Predictive values for the absence of UTI (absence of ≥1 of the following criteria: documentation of UTI diagnosis, antibiotic prescription, uropathogen presence on culture) were calculated. RESULTS: In total, 22 883 UAs were included. Negative UA had a high predictive value for negative urine culture (0.95, 95% confidence interval [CI]: 0.94-0.95) and absence of UTI (0.99, CI: 0.99-0.995) in the overall cohort. Negative UA also had a high predictive value for negative urine culture (0.93, CI: 0.90-0.95) and absence of UTI (0.99, CI: 0.98-0.999) in those with indwelling urinary catheters. The traditional threshold of culture growth of 100 000 colony-forming units (CFU)/mL did not capture 22% of UTIs. CONCLUSION: UA exhibits high predictive value for negative urine culture and absence of UTI in men, supporting a protocol wherein culture is only performed in the context of abnormal UA. The traditional 100 000 CFU/mL cut-off may have not captured a subset of UTI in the male population, and warrants further investigation.

Sacral neuromodulation device biofilm differs in the absence and presence of infection, harbors antibiotic resistance genes, and is reproducible in vitro.

INTRODUCTION/PURPOSE: Sacral neuromodulation (SNM) is effective therapy for overactive bladder refractory to oral therapies, and non-obstructive urinary retention. A subset of SNM devices is associated with infection requiring surgical removal. We sought to compare microbial compositions of explanted devices in the presence and absence of infection, by testing phase, and other clinical factors, and to investigate antibiotic resistance genes present in the biofilms. We analyzed resistance genes to antibiotics used in commercially-available anti-infective device coating/pouch formulations. We further sought to assess biofilm reconstitution by material type and microbial strain in vitro using a continuous-flow stir tank bioreactor, which mimics human tissue with an indwelling device. We hypothesized that SNM device biofilms would differ in composition by infection status, and genes encoding resistance to rifampin and minocycline would be frequently detected. MATERIALS/METHODS: Patients scheduled to undergo removal or revision of SNM devices were consented per IRB-approved protocol (IRB 20-415). Devices were swabbed intraoperatively upon exposure, with controls and precautions to reduce contamination of the surrounding field. Samples and controls were analyzed with next-generation sequencing and RT-PCR, metabolomics, and culture-based approaches. Associations between microbial diversity or microbial abundance, and clinical variables were then analyzed using t-tests and ANOVA. Reconstituted biofilm deposition in vitro using the bioreactor was compared by microbial strain and material type using plate-based assays and scanning electron microscopy. RESULTS: Thirty seven devices were analyzed, all of which harbored detectable microbiota. Proteobacteria, Firmicutes and Actinobacteriota were the most common phyla present overall. Beta-diversity differed in the presence versus absence of infection (p = 0.014). Total abundance, based on normalized microbial counts, differed by testing phase (p < 0.001), indication for placement (p = 0.02), diabetes mellitus (p < 0.001), cardiac disease (p = 0.008) and history of UTI (p = 0.008). Significant microbe-metabolite interaction networks were identified overall and in the absence of infection. 24% of biofilms harbored the tetA tetracycline/minocycline resistance gene and 53% harbored the rpoB rifampin resistance gene. Biofilm was reconstituted across tested strains and material types. Ceramic and titanium did not differ in biofilm deposition for any tested strain. CONCLUSIONS: All analyzed SNM devices harbored microbiota. Device biofilm composition differed in the presence and absence of infection and by testing phase. Antibiotic resistance genes including to rifampin and tetracycline/minocycline, which are used in commercially-available anti-infective pouches, were frequently detected. Isolated organisms from SNM devices demonstrated the ability to reconstitute biofilm formation in vitro. Biofilm deposition was similar between ceramic and titanium, materials used in commercially-available SNM device casings. The findings and techniques used in this study together provide the basis for the investigation of the next generation of device materials and coatings, which may employ novel alternatives to traditional antibiotics. Such alternatives might include bacterial competition, quorum-sensing modulation, or antiseptic application, which could reduce infection risk without significantly selecting for antibiotic resistance.

How Can We Show That Artificial Intelligence May Improve Our Assessment and Management of Lower Urinary Tract Dysfunctions?-ICI-RS 2024.

AIMS: The integration of artificial intelligence (AI) into functional urology management must be assessed for its clinical utility, but hopefully will change, perhaps to revolutionize the way LUTD and other conditions are assessed, the aim being to offer patients more rapid and effective management which enhances patient outcomes. The aim of this proposal, discussed at the ICI-RS annual meeting, is to evaluate the available evidence on AI and the way it might change the approach to urodynamic (UDS) diagnoses, including overactive bladder syndrome (OAB), and perhaps other LUTDs such as bladder outflow obstruction. METHODS: A compendium of discussion based on the current evidence related to AI and its potential applications in UDS and OAB. RESULTS: AI-powered diagnostic tools are being developed to analyze complex datasets from urodynamic studies, imaging, and other diagnostic tests. AI systems can leverage large volumes of clinical data to recommend personalized treatment plans based on individual patient profiles to optimize surgical procedures, enhance diagnostic precision, tailor the therapy, reduce the risk of complications, and improve outcomes. In the future, AI will be able to provide tailored counseling regarding the outcomes and potential side effects of drugs and procedures to a given patient. CONCLUSION: AI's role in functional urology has been poorly investigated, and its implementation across several areas may improve clinical care and the pathophysiological understanding of functional urologic conditions.

Handover mechanism of the growing pilus by the bacterial outer-membrane usher FimD.

Pathogenic bacteria such as Escherichia coli assemble surface structures termed pili, or fimbriae, to mediate binding to host-cell receptors1. Type 1 pili are assembled via the conserved chaperone-usher pathway2-5. The outer-membrane usher FimD recruits pilus subunits bound by the chaperone FimC via the periplasmic N-terminal domain of the usher. Subunit translocation through the ß-barrel channel of the usher occurs at the two C-terminal domains (which we label CTD1 and CTD2) of this protein. How the chaperone-subunit complex bound to the N-terminal domain is handed over to the C-terminal domains, as well as the timing of subunit polymerization into the growing pilus, have previously been unclear. Here we use cryo-electron microscopy to capture a pilus assembly intermediate (FimD-FimC-FimF-FimG-FimH) in a conformation in which FimD is in the process of handing over the chaperone-bound end of the growing pilus to the C-terminal domains. In this structure, FimF has already polymerized with FimG, and the N-terminal domain of FimD swings over to bind CTD2; the N-terminal domain maintains contact with FimC-FimF, while at the same time permitting access to the C-terminal domains. FimD has an intrinsically disordered N-terminal tail that precedes the N-terminal domain. This N-terminal tail folds into a helical motif upon recruiting the FimC-subunit complex, but reorganizes into a loop to bind CTD2 during handover. Because both the N-terminal and C-terminal domains of FimD are bound to the end of the growing pilus, the structure further suggests a mechanism for stabilizing the assembly intermediate to prevent the pilus fibre diffusing away during the incorporation of thousands of subunits.

Estimated Glomerular Filtration Rate Using Cystatin C is a More Sensitive Marker for Kidney Dysfunction in Nonweight-bearing Individuals.

PURPOSE: Individuals with neuromuscular disorders and neurogenic lower urinary tract dysfunction are commonly nonweight-bearing with lower lean muscle mass than the general population. We sought to compare estimated glomerular filtration rate equations that include creatinine, cystatin C, or both, in nonweight-bearing individuals and matched ambulatory controls. MATERIALS AND METHODS: Records were reviewed for individuals with serum creatinine (Cr) and cystatin C (Cys) and diagnosis consistent with nonweight-bearing status, and matched ambulatory controls. The 2021 CKD-EPI (Chronic Kidney Disease-Epidemiology Collaboration) race agnostic equations were used to calculate estimated glomerular filtration rate. Renal function was compared by equation in the overall cohorts and in a patient subset with imaging and/or urinalysis evidence of renal dysfunction. RESULTS: Nonweight-bearing (n = 102) and control populations (n = 204) had similar demographics. In the nonweight-bearing population, estimated glomerular filtration rate differed when calculated using CKD-EPICr, CKD-EPICr+Cys, and CKD-EPICys (107, 93, 80 mL/min/1.73 m2, respectively, P < .001). The differences in estimated glomerular filtration rate were greater in the nonweight-bearing relative to the control group regardless of CKD-EPI equation pairs compared (P < .001). In the patient subset with imaging and/or proteinuria evidence of renal dysfunction, the nonweight-bearing population again had different estimated glomerular filtration rate when calculated using CKD-EPICr, CKD-EPICr+Cys, and CKD-EPICys (P < .001). Fifty-eight percent of nonweight-bearing individuals with evidence of renal dysfunction on imaging or urinalysis were reclassified into a lower estimated glomerular filtration rate category when using estimated glomerular filtration rateCys relative to estimated glomerular filtration rateCr. CONCLUSIONS: Estimated glomerular filtration rate equations containing serum creatinine, cystatin C, or both, validated in mostly ambulatory populations, are not equivalently accurate in estimating kidney function in nonweight-bearing individuals. Comparison of these equations against gold standard glomerular filtration rate measurement is needed to determine which most closely approximates true glomerular filtration rate.

Ureteral Stents Harbor Complex Biofilms With Rich Microbiome-Metabolite Interactions.

PURPOSE: We sought to determine microbe-metabolite composition and interactions within indwelling ureteral stent biofilms, determine their association with patient factors including infection, and reconstitute biofilm formation on relevant surface materials in vitro. MATERIALS AND METHODS: Upon ureteral stent removal from patients, proximal and distal ends were swabbed. Samples were analyzed by 16S next-generation sequencing and metabolomics. A continuous-flow stir-tank bioreactor was used to reconstitute and quantify in vitro biofilm formation from stent-isolated bacteria on stent-related materials including silicone, polytetrafluoroethylene, polyurethane, polycarbonate, and titanium. Diversity, relative abundance, and association with clinical factors were analyzed with ANOVA and Bonferroni t-tests or PERMANOVA. Biofilm deposition by microbial strain and device material type were analyzed using plate counts and scanning electron microscopy following bioreactor incubation. RESULTS: All 73 samples from 37 ureteral stents harbored microbiota. Specific genera were more abundant in samples from stents wherein there was antibiotic exposure during indwelling time (Escherichia/Shigella, Pseudomonas, Staphylococcus, Ureaplasma) and in those associated with infection (Escherichia/Shigella, Ureaplasma). The enriched interaction subnetwork in stent-associated infection included Ureaplasma and metabolite 9-methyl-7-bromoeudistomin. Strains identified as clinically relevant and central to interaction networks all reconstituted biofilm in vitro, with differential formation by strain (Enterococcus faecalis most) and material type (titanium least). CONCLUSIONS: Ureteral stent biofilms exhibit patterns unique to stent-associated infection and antibiotic exposure during indwelling time. Microbes isolated from stents reconstituted biofilm formation in vitro. This work provides a platform to test novel materials, evaluate new coatings for anti-biofilm properties, and explore commensal strain use for bacterial interference against pathogens.

Penile prostheses harbor biofilms driven by individual variability and manufacturer even in the absence of clinical infection.

BACKGROUND: Culture-based studies have shown that penile prostheses harbor biofilms in the presence and absence of infection, but these findings have not been adequately validated using contemporary microbiome analytic techniques. AIM: The study sought to characterize microbial biofilms of indwelling penile prosthesis devices according to patient factors, device components, manufacturer, and infection status. METHODS: Upon penile prostheses surgical explantation, device biofilms were extracted, sonicated, and characterized using shotgun metagenomics and culture-based approaches. Device components were also analyzed using scanning electron microscopy. OUTCOMES: Outcomes included the presence or absence of biofilms, alpha and beta diversity, specific microbes identified and the presence of biofilm, and antibiotic resistance genes on each prosthesis component. RESULTS: The average age of participants from whom devices were explanted was 61 ± 11 years, and 9 (45%) of 20 had a diagnosis of diabetes mellitus. Seventeen devices were noninfected, and 3 were associated with clinical infection. Mean device indwelling time prior to explant was 5.1 ± 5.1 years. All analyzed components from 20 devices had detectable microbial biofilms, both in the presence and absence of infection. Scanning electron microscopy corroborated the presence of biofilms across device components. Significant differences between viruses, prokaryotes, and metabolic pathways were identified between individual patients, device manufacturers, and infection status. Mobiluncus curtisii was enriched in manufacturer A device biofilms relative to manufacturer B device biofilms. Bordetella bronchialis, Methylomicrobium alcaliphilum, Pseudoxanthomonas suwonensis, and Porphyrobacter sp. were enriched in manufacturer B devices relative to manufacturer A devices. The most abundant bacterial phyla were the Proteobacteria, Actinobacteria, and Firmicutes. Glycogenesis, the process of glycogen synthesis, was among the predominant metabolic pathways detected across device components. Beta diversity of bacteria, viruses, protozoa, and pathways did not differ among device components. CLINICAL IMPLICATIONS: All components of all penile prostheses removed from infected and noninfected patients have biofilms. The significance of biofilms on noninfected devices remains unknown and merits further investigation. STRENGTHS AND LIMITATIONS: Strengths include the multipronged approach to characterize biofilms and being the first study to include all components of penile prostheses in tandem. Limitations include the relatively few number of infected devices in the series, a relatively small subset of devices included in shotgun metagenomics analysis, and the lack of anaerobic and other expanded conditions for culture. CONCLUSION: Penile prosthesis biofilms are apparent in the presence and absence of infection, and the composition of biofilms was driven primarily by device manufacturer, individual variability, and infection, while being less impacted by device component.

Neural networks outperform expert humans in predicting patient impressions of symptomatic improvement following overactive bladder treatment.

INTRODUCTION AND HYPOTHESIS: The objective was to accurately predict patient-centered subjective outcomes following the overactive bladder (OAB) treatments OnabotulinumtoxinA (OBTX-A) injection and sacral neuromodulation (SNM) using a neural network-based machine-learning approach. In the context of treatments designed to improve quality of life, a patient's perception of improvement should be the gold standard outcome measure. METHODS: Cutting-edge neural network-based algorithms using reproducing kernel techniques were trained to predict patient-reported improvements in urinary leakage and bladder function as assessed by Patient Global Impression of Improvement score using the ROSETTA trial datasets. Blinded expert urologists provided with the same variables also predicted outcomes. Receiver operating characteristic curves and areas under the curve were generated for algorithm and human expert predictions in an out-of-sample holdout dataset. RESULTS: Algorithms demonstrated excellent accuracy in predicting patient subjective improvement in urinary leakage (OBTX-A: AUC 0.75; SNM: 0.80). Similarly, algorithms accurately predicted patient subjective improvement in bladder function (OBTX-A: AUC 0.86; SNM: 0.96). The top-performing algorithms outcompeted human experts across outcome measures. CONCLUSIONS: Novel neural network-based machine-learning algorithms accurately predicted OBTX-A and SNM patient subjective outcomes, and generally outcompeted expert humans. Subtle aspects of the physician-patient interaction remain uncomputable, and thus the machine-learning approach may serve as an aid, rather than as an alternative, to human interaction and clinical judgment.

Machine learning provides an accurate prognostication model for refractory overactive bladder treatment response and is noninferior to human experts.

OBJECTIVE: The increasing wealth of clinical data may become unmanageable for a physician to assimilate into optimal decision-making without assistance. Utilizing a novel machine learning (ML) approach, we sought to develop algorithms to predict patient outcomes following the overactive bladder treatments OnabotulinumtoxinA (OBTX-A) injection and sacral neuromodulation (SNM). MATERIALS AND METHODS: ROSETTA datasets for overactive bladder patients randomized to OBTX-A or SNM were obtained. Novel ML algorithms, using reproducing kernel techniques were developed and tasked to predict outcomes including treatment response and decrease in urge urinary incontinence episodes in both the OBTX-A and SNM cohorts, in validation and test sets. Blinded expert urologists also predicted outcomes. Receiver operating characteristic curves were generated and AUCs calculated for comparison to lines of ignorance and the expert urologists' predictions. RESULTS: Trained algorithms demonstrated outstanding accuracy in predicting treatment response (OBTX-A: AUC 0.95; SNM: 0.88). Algorithms accurately predicted mean decrease in urge urinary incontinence episodes (MSE < 0.15) in OBTX-A and SNM. Algorithms were superior to human experts in response prediction for OBTX-A, and noninferior to human experts in response prediction for SNM. CONCLUSIONS: Novel ML algorithms were accurate, superior to expert urologists in predicting OBTX-A outcomes, and noninferior to expert urologists in predicting SNM outcomes. Some aspects of the physician-patient interaction are subtle and uncomputable, and thus ML may complement, but not supplant, a physician's judgment.

Same-day discharge across FPMRS surgical cases is safe and feasible: A 10-year single-surgeon experience.

INTRODUCTION: Enhanced recovery after surgery protocols and increased attention to value-based care have led to the reconsideration of routine postoperative admission in female pelvic medicine and reconstructive surgery (FPMRS) cases. We aimed to assess trends in same-day discharge (SDD) and associated readmissions and emergency room visits in a single-surgeon 10-year experience. METHODS: The electronic medical record was queried for 30-day outcomes (readmission and emergency department visits with associated indications) for all cases performed between June 2010 and August 2020 by a single FPMRS surgeon. Non-FPMRS specialty cases were excluded. Patient characteristics and 30-day outcomes were compared based on SDD status for the overall cohort as well as the subset of cases traditionally involving an overnight stay (i.e., robotic transabdominal, apical prolapse repair). RESULTS: 1793 surgeries were identified and analyzed, including 357 apical prolapse repairs, 370 slings, 392 neuromodulation and 114 complex mesh excisions. The majority (79.1%) had SDD. For admitted patients, mean length of stay was 1.5 (1.3) days. Among cases traditionally involving overnight stay, rates of SDD were significantly higher in 2020 than 2010 (84% vs. 32%, p < 0.001), and increased over time. Overall rates of 30-day readmission and ED visits were low (1.9% and 2.6%, respectively) and did not differ based on SDD status (p = 0.76). Readmissions occurred at mean 11.6 (7.0) days, most commonly for urinary tract infection (13/34). CONCLUSIONS: SDD is not associated with increased 30-day readmission or ED visits across a wide breadth of FPMRS cases. SDD is safe and feasible in the majority of FPMRS cases.

The Natural History and Composition of Urinary Catheter Biofilms: Early Uropathogen Colonization with Intraluminal and Distal Predominance.

PURPOSE: We sought to determine the composition and initiation site of bacterial biofilm on indwelling urinary catheters and to track biofilm progression with time. MATERIALS AND METHODS: Indwelling urinary catheters were collected from 2 tertiary care centers following removal from patients. Indwelling time was noted and catheters were de-identified. Catheters were sectioned, stained for biofilms and analyzed by spectrophotometry and visualization. Biofilm colonization patterns were analyzed using descriptive statistical analysis and bacterial composition was determined using next generation sequencing. RESULTS: We collected and analyzed a total of 33 catheters from 26 males and 7 females with indwelling time ranging from 15 minutes to 43 days. Biofilm colonization was consistently high on the region of the balloon for all indwelling times. After week 1 the distal third of the catheter had higher biofilm colonization than the proximal third (week 2 p=0.034). At all indwelling times the intraluminal surface of the catheter had greater biofilm colonization than the outer surface. Next generation sequencing detected potential uropathogenic bacteria in all 10 analyzed samples. CONCLUSIONS: The catheter balloon, its distal aspect and its lumen were the predominant locations of biofilm comprising uropathogenic bacteria. Strategies to prevent or treat biofilm should be targeted to these areas.

PSA kinetics following primary focal cryotherapy (hemiablation) in organ-confined prostate cancer patients.

PURPOSE: We aim to evaluate prostate-specific antigen (PSA) trends in post-primary focal cryotherapy (PFC) patients. MATERIALS AND METHODS: This was an institutional review board-approved retrospective study of PFC patients from 2010 to 2015. Patients with at least one post-PFC PSA were included in the study. Biochemical recurrence (BCR) was determined using the Phoenix criteria. PSA bounce was also assessed. We analyzed rates of change of PSA over time of post-PFC between BCR and no BCR groups. PSA-derived variables were analyzed as potential predictors of BCR. RESULTS: A total of 104 PFC patients were included in our analysis. Median (range) age and follow-up time were 66 (48-82) years and 19 (6.3-38.6) months, respectively. Four (3.8%) patients experienced PSA bounce. The median percent drop in first post-PFC PSA of 80.0% was not associated with BCR (p = 0.256) and may indicate elimination of the index lesion. The rate of increase of PSA in BCR patients was significantly higher compared to patients who did not recur (median PSA velocity (PSAV): 0.15 vs 0.04 ng/ml/month, p = 0.001). Similar to PSAV (HR 9.570, 95% CI 3.725-24.592, p < 0.0001), PSA nadir ≥ 2 ng/ml [HR (hazard ratio) 1.251, 95% CI 1.100-1.422, p = 0.001] was independently associated with BCR. CONCLUSION: A significant drop in post-PFC PSA may indicate elimination of the index lesion. Patients who are likely to recur biochemically have a significantly higher PSAV compared to those who do not recur. Nadir PSA of less than 2 ng/ml may be considered the new normal PSA in focal cryotherapy (hemiablation) follow-up.

The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution.

UNLABELLED: The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains of Escherichia coli use this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in the periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 ß-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function. IMPORTANCE: Pili are critical virulence factors for many bacterial pathogens. Uropathogenic E. coli relies on P and type 1 pili assembled by the chaperone/usher pathway to adhere to the urinary tract and establish infection. Studying pilus assembly is important for understanding mechanisms of protein secretion, as well as for identifying points for therapeutic intervention. Pilus biogenesis is a multistep process. This work investigates the oligomeric state of the pilus chaperone in the periplasm, which is important for understanding early assembly events. Our work unambiguously demonstrates that both PapD and FimC chaperones are monomeric in solution. We further demonstrate that the solution behavior of the FimH and PapGII adhesins differ, which may be related to functional differences between the two pilus systems.

Identification and characterization of peptidases secreted by quahog parasite unknown (QPX), the protistan parasite of hard clams.

Quahog parasite unknown (QPX) is a protistan parasite capable of causing deadly infections in the hard clam Mercenaria mercenaria, one of the most valuable shellfish species in the USA. QPX is an extracellular parasite found mostly in the connective tissue of clam mantle and, in more severe cases of infection, other clam organs. Histopathologic examinations revealed that QPX cells within clam tissues are typically surrounded by hollow areas that have been hypothesized to be, at least in part, a result of extracellular digestion of clam proteins by the parasite. We investigated peptidase activity in QPX extracellular secretions using sodium dodecyl sulfate-polyacrylamide gels containing gelatin as a co-polymerized substrate. Multiple peptidase activity bands of molecular weights ranging from 20 to 100 kDa were detected in QPX secretions derived from a variety of culture media. One major band of approximately 35 kDa was composed of subtilisin-like peptidases that were released by QPX cells in all studied media, suggesting that these are the most common peptidases used by QPX for nutrient acquisition. PCR quantification of mRNA encoding QPX subtilisins revealed that their expression changes with the protein substrate used in the culture media. A fast protein liquid chromatography (FPLC) was used to fractionate QPX extracellular secretions. An FPLC-fraction containing a subtilisin-type serine peptidase was able to digest clam plasma proteins, suggesting that this peptidase might be involved in the disease process, and making it a good candidate for further investigation as a possible virulence factor of the parasite.

Electrostatic networks control plug stabilization in the PapC usher.

The PapC usher, a ß-barrel pore in the outer membrane of uropathogenic Escherichia coli, is used for assembly of the P pilus, a key virulence factor in bacterial colonization of human kidney cells. Each PapC protein is composed of a 24-stranded ß-barrel channel, flanked by N- and C-terminal globular domains protruding into the periplasm, and occluded by a plug domain (PD). The PD is displaced from the channel towards the periplasm during pilus biogenesis, but the molecular mechanism for PD displacement remains unclear. Two structural features within the ß-barrel, an α-helix and ß5-6 hairpin loop, may play roles in controlling plug stabilization. Here we have tested clusters of residues at the interface of the plug, barrel, α-helix and hairpin, which participate in electrostatic networks. To assess the roles of these residues in plug stabilization, we used patch-clamp electrophysiology to compare the activity of wild-type and mutant PapC channels containing alanine substitutions at these sites. Mutations interrupting each of two salt bridge networks were relatively ineffective in disrupting plug stabilization. However, mutation of two pairs of arginines located at the inner and the outer surfaces of the PD resulted in an enhanced propensity for plug displacement. One arginine pair involved in a repulsive interaction between the linkers that tether the plug to the ß-barrel was particularly sensitive to mutation. These results suggest that plug displacement, which is necessary for pilus assembly and translocation, may require a weakening of key electrostatic interactions between the plug linkers, and the plug and the α-helix.