Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat due to multi-drug resistance development among the CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, we found that E. faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Axotomy results in an increase in Thy-1 protein in the 35-day-old rat supraoptic nucleus.

We demonstrated previously that the hypothalamic supraoptic nucleus (SON) undergoes an axonal sprouting response following a unilateral lesion of the hypothalamo-neurohypophysial tract in a 35-day-old rat to repopulate the partially denervated neural lobe (NL). However, no sprouting occurs following the same injury in a 125-day-old rat. We previously reported a significant increase in Thy-1 protein in the SON of a 125-day-old rat compared to a 35-day-old rat in the absence of injury. Thy-1 is a cell surface glycoprotein shown to inhibit axonal outgrowth following injury; however, we did not look at axotomy's effect on Thy-1 in the SON. Therefore, we sought to determine the integrin ligands that bind Thy-1 in the SON and how axotomy impacts Thy-1. Like what others have shown, the co-immunoprecipitation analysis demonstrated that Thy-1 interacts with αvß3 and αvß5 integrin dimers in the SON. We used western blot analysis to examine protein levels of Thy-1 and integrin subunits following injury in the 35- and 125-day-old rat SON and NL. Our results demonstrated that Thy-1 protein levels increase in the lesion SON in a 35-day-old rat. The quantitative dual-fluorescent analysis showed that the increase in Thy-1 in the lesion SON occurred in astrocytes. There was no change in Thy-1 or integrin protein levels following injury in the 125-day-old following injury. Furthermore, the axotomy significantly decreased Thy-1 protein levels in the NL of both 35- and 125-day-old rats. These results provide evidence that Thy-1 protein levels are injury dependent in the magnocellular neurosecretory system.

c-di-AMP Is Essential for the Virulence of Enterococcus faecalis.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

Enterococcus faecalis Polymicrobial Interactions Facilitate Biofilm Formation, Antibiotic Recalcitrance, and Persistent Colonization of the Catheterized Urinary Tract.

Indwelling urinary catheters are common in health care settings and can lead to catheter-associated urinary tract infection (CAUTI). Long-term catheterization causes polymicrobial colonization of the catheter and urine, for which the clinical significance is poorly understood. Through prospective assessment of catheter urine colonization, we identified Enterococcus faecalis and Proteus mirabilis as the most prevalent and persistent co-colonizers. Clinical isolates of both species successfully co-colonized in a murine model of CAUTI, and they were observed to co-localize on catheter biofilms during infection. We further demonstrate that P. mirabilis preferentially adheres to E. faecalis during biofilm formation, and that contact-dependent interactions between E. faecalis and P. mirabilis facilitate establishment of a robust biofilm architecture that enhances antimicrobial resistance for both species. E. faecalis may therefore act as a pioneer species on urinary catheters, establishing an ideal surface for persistent colonization by more traditional pathogens such as P. mirabilis.

Age-dependent increase in Thy-1 protein in the rat supraoptic nucleus.

Mature mammalian CNS neurons often do not recover successfully following injury. To this point, unilateral lesion of the hypothalamo-neurohypophysial tract results in collateral sprouting from uninjured axons of the supraoptic nucleus (SON) in 35-day-old but not in 125-day-old rats. Thus, it appears that there are age-related changes within the SON that preclude the older rat from recovering following axotomy. We hypothesize that the intrinsic capacity for axon reorganization may depend, in part, on age-related alterations in cell adhesion molecules that allow normal astrocyte-neuron interactions in the SON. In support of our hypothesis, numerous reports have shown that Thy-1 is increased in neurons at the cessation of axon outgrowth. Therefore, we compared protein levels of Thy-1 and the Thy-1 interacting integrin subunits, alpha-v (αv), beta-3 (ß3), and beta-5 (ß5), in 35- and 125-day-old SON using western blot analysis. Our results demonstrated that there was significantly more Thy-1 protein in the 125-day-old SON compared to 35-day-old SON, but no change in the protein levels of the integrin subunits. Furthermore, we localized Thy-1-, αv integrin-, ß3 integrin-, and ß5 integrin-immunoreactivity to both neurons and astrocytes in the SON. Altogether, our results suggest that the observed increase in Thy-1 protein levels in the SON with age may contribute to an environment that prevents collateral axonal sprouting in the SON of the 125-day-old rat.