Jerome S. Pinkner

Catheter-associated urinary tract infections (CAUTIs) account for approximately 80% of urinary tract infections (UTI) and can lead to adverse outcomes. Most CAUTIs are polymicrobial with resilient communities maintaining a consistent composition of species over time. However, the mechanisms promoting persistence are poorly understood. Here, we examine how a chemical interaction between Enterococcus faecalis and Klebsiella pneumoniae can explain their high rate of co-occurrence on long-term indwelling catheters. Sequence analyses of longitudinal isolates from several patients coinfected with E. faecalis and K. pneumoniae revealed that despite frequent replacement, catheters became recolonized with the same or a nearly identical consortium of strains throughout the collection period. Using artificial urine medium (AUM), monoculture revealed that the K. pneumoniae isolates grew robustly and formed biofilm, while the E. faecalis isolates grew poorly and did not form biofilm. However, coculture of paired isolates resulted in enhanced E. faecalis growth and biofilm, which could be reproduced by supplementing E. faecalis with K. pneumoniae conditioned AUM supernatant (KpAUMSup). Analyses using comparative transcriptomics, mutant strains, and chemical inhibitors with cell culture and murine CAUTI models revealed that (i) KpAUMSup, but not AUM, stimulated expression of the E. faecalis Fsr quorum sensing system; (ii) Fsr was required for E. faecalis to respond to KpAUMSup; (iii) E. faecalis cultured in KpAUMSup was more efficient in initiating CAUTI; and (iv) disruption of Fsr inhibited initiation of CAUTI. This interspecies signaling may help explain the high rate of co-colonization of these CAUTI pathogens and highlights therapeutic strategies to treat polymicrobial CAUTI.

Antimicrobial resistance (AMR) in common bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), is an increasingly dire public health threat, with MRSA accounting for up to 90% of S. aureus infections. To expand the treatment arsenal against MRSA infections, we developed a class of tunable three-dimensional tricyclic 2-pyridones, termed TriPcides, that can kill MRSA resistant to last-resort antibiotics and eliminate MRSA persister cells. No preexisting resistance was detected across hundreds of clinical isolates, and continuous exposure of MRSA to TriPcides did not elicit detectable resistance. Treatment with TriPcides causes a rapid decrease in membrane integrity and increased levels of reactive oxygen species. Last, TriPcides effectively reduce secretion of important virulence factors and result in reduced ulcer size and healing time in S. aureus murine skin and soft tissue infections but do not reduce bacterial burden.

The Yeh chaperone-usher pathway (CUP) pilus adhesin is encoded in one-half of all Escherichia coli . Yet little is known about its structure and function in E. coli persistence and pathogenesis. Structural investigations reveal that the adhesin receptor binding domains (RBDs) of YehD and its relative YhlD both share a canonical β-rich core and an α-helical flap motif that is hinged at the distal end of the core. This flap was observed in both open and closed conformations using molecular dynamics simulations. The closed conformation is dependent on a hydrophobic patch of amino acids on the distal end of the flap. Functionally, YehD _RBD is able to bind pectin, a polysaccharide ubiquitous in plant material. Mutations that interrupt the closed conformation increase the affinity of the protein to pectin, suggesting that the flap contributes mechanistically to pectin binding. Furthermore, in vivo, the pilus contributes to gastrointestinal (GI) tract colonization in the absence of the type 1 pilus. Hence, we report the ability of YehD to bind pectin representing a possible colonization mechanism of the GI tract via a structurally distinct CUP adhesin.

The rise of multidrug-resistant bacterial infections necessitates the discovery of novel antimicrobial strategies. Here, we show that protein design provides a generalizable means of generating new antimicrobials by neutralizing the function of bacterial adhesins, which are virulence factors critical in host-pathogen interactions. We de novo designed high-affinity miniprotein binders to FimH and Abp1D/Abp2D chaperone usher pili adhesins from uropathogenic Escherichia coli and Acinetobacter baumannii , respectively, which are implicated in mediating both uncomplicated and catheter-associated urinary tract infections (UTI) responsible for significant morbidity and mortality worldwide. The designed antagonists have high specificity and stability, disrupt bacterial recognition of host receptors, block biofilm formation, and are effective in treating and preventing uncomplicated and catheter-associated UTIs in vivo . Targeting virulence factors outside the cell membrane with protein design provides a rapid route to next-generation therapeutics that can disrupt pathogenesis and thus are capable of treating and preventing disease in an antibiotic-sparing manner.

As antimicrobial resistance increases, urinary tract infections (UTIs) are expected to pose an increased burden in morbidity and expense on the health care system, increasing the need for alternative antibiotic-sparing treatments. Most UTIs are caused by uropathogenic Escherichia coli (UPEC), whereas Klebsiella pneumoniae causes a large portion of non-UPEC UTIs. Both bacteria express type 1 pili tipped with the mannose-binding FimH adhesin critical for UTI pathogenesis. We generated and biochemically characterized 33 murine monoclonal antibodies (mAbs) to FimH. Three mAbs protected mice from E. coli UTI. Mechanistically, we show that this protection is Fc independent and mediated by the ability of these mAbs to sterically block FimH function by recognizing a high-affinity FimH conformation. Our data reveal that FimH mAbs hold promise as an antibiotic-sparing treatment strategy.

Urinary tract infections (UTIs) are highly recurrent and frequently caused by Uropathogenic Escherichia coli (UPEC) strains that can be found in patient intestines. Seeding of the urinary tract from this intestinal reservoir likely contributes to UTI recurrence (rUTI) rates. Thus, understanding the factors that promote UPEC intestinal colonization is of critical importance to designing therapeutics to reduce rUTI incidence. Although E. coli is found in high abundance in large intestine mucus, little is known about how it is able to maintain residence in this continuously secreted hydrogel. We discovered that the FimH adhesin of type 1 pili (T1P) bound throughout the secreted mucus layers of the colon and to epithelial cells in mouse and human samples. Disruption of T1P led to reduced association with colon mucus. Notably, this mutant up-regulated flagellar production and infiltrated the protective inner mucus layer of the colon. This could explain how UPEC resists being washed off by the continuously secreted mucus layers of the colon.

We have developed GmPcides from a peptidomimetic dihydrothiazolo ring-fused 2-pyridone scaffold that has antimicrobial activities against a broad spectrum of Gram-positive pathogens. Here, we examine the treatment efficacy of GmPcides using skin and soft tissue infection (SSTI) and biofilm formation models by Streptococcus pyogenes . Screening our compound library for minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations identified GmPcide PS757 as highly active against S. pyogenes . Treatment of S. pyogenes biofilm with PS757 revealed robust efficacy against all phases of biofilm formation by preventing initial biofilm development, ceasing biofilm maturation and eradicating mature biofilm. In a murine model of S. pyogenes SSTI, subcutaneous delivery of PS757 resulted in reduced levels of tissue damage, decreased bacterial burdens, and accelerated rates of wound healing, which were associated with down-regulation of key virulence factors, including M protein and the SpeB cysteine protease. These data demonstrate that GmPcides show considerable promise for treating S. pyogenes infections.

Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenicEscherichia coli(UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed anin vitromodel of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimHin vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis.

Though rare, breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), a CD30+ T-cell lymphoma associated with textured breast implants, has adversely impacted our perception of the safety of breast implants. Its etiology unknown, one hypothesis suggests an initiating inflammatory stimulus, possibly infectious, triggers BIA-ALCL. We analyzed microbiota of breast, skin, implant and capsule in BIA-ALCL patients (n = 7), and controls via culturing methods, 16S rRNA microbiome sequencing, and immunohistochemistry. Alpha and beta diversity metrics and relative abundance of Gram-negative bacteria were calculated, and phylogenetic trees constructed. Staphylococcus spp., the most commonly cultured microbes, were identified in both the BIA-ALCL and contralateral control breast. The diversity of bacterial microbiota did not differ significantly between BIA-ALCL and controls for any material analyzed. Further, there were no significant differences in the relative abundance of Gram-negative bacteria between BIA-ALCL and control specimens. Heat maps suggested substantial diversity in the composition of the bacterial microbiota of the skin, breast, implant and capsule between patients with no clear trend to distinguish BIA-ALCL from controls. While we identified no consistent differences between patients with BIA-ALCL-affected and contralateral control breasts, this study provides insights into the composition of the breast microbiota in this population.

Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-β and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors. Urinary tract infections affect millions of individuals annually, and many patients suffer from recurring infections several times a year. Antibiotic resistance is increasing rapidly and new strategies are needed to treat even these common bacterial infections. One approach is to use the protective power of asymptomatic bacterial carriage, which has been shown to protect the host against symptomatic urinary tract infection. Instilling “nice” bacteria in the urinary bladder is therefore a promising alternative approach to antibiotic therapy. In an effort to increase the therapeutic use of asymptomatic bacteriuria, we reintroduced bacterial adhesion molecules into the therapeutic Escherichia coli strain 83972 and inoculated patients who are in need of alternative therapy. To our great surprise, the P fimbriated variant caused symptoms, despite lacking other virulence factors commonly thought to be necessary to cause disease. In contrast, type 1 fimbriae, did not provoke symptoms but enhanced the beneficial properties of the wild-type strain. This is explained by a divergent effect of these fimbrial types on host gene expression, where P fimbriae activate the IRF-7 transcription factor that regulates pathology in infected kidneys, suggesting that a single, potent virulence gene may be sufficient to create virulence in human hosts.