Cell surface hydrophobicity (CSH) is an important cellular biophysical parameter which affects both cell-cell and cell-surface interactions. In dimorphic fungi, multiple factors including the ...temperature-induced shift between mold and yeast forms have strong effects on CSH with higher hydrophobicity more common at the lower temperatures conducive to filamentous cell growth. Some strains of
exhibit high CSH despite the presence of the hydrophilic capsule. Among individual yeast colonies from the same isolate, distinct morphologies can correspond to differences in CSH. These differences in CSH are frequently associated with altered virulence in medically-significant fungi and can impact the efficacy of antifungal therapies. The mechanisms for the maintenance of CSH in pathogenic fungi remain poorly understood, but an appreciation of this fundamental cellular parameter is important for understanding its contributions to such phenomena as biofilm formation and virulence.
The stringent response is well conserved across bacterial species and is a key pathway involved both in bacterial survival and virulence and in the induction of antibiotic tolerance in Mycobacteria. ...It is mediated by the alarmone (p)ppGpp and the regulatory molecule inorganic polyphosphate in response to stress conditions such as nutrient starvation. Efforts to pharmacologically target various components of the stringent response have shown promise in modulating mycobacterial virulence and antibiotic tolerance. In this review, we summarize the current understanding of the stringent response and its role in virulence and tolerance in Mycobacteria, including evidence that targeting this pathway could have therapeutic benefit.
and
are pathogenic fungi that cause significant morbidity and mortality. Cell surface hydrophobicity (CSH) is a biophysical parameter that influences the adhesion of fungal cells or spores to biotic ...and abiotic surfaces.
is encased by polysaccharide capsule that is highly hydrophilic and is a critical determinant of virulence. In this study, we report large differences in the CSH of some
and
strains. The capsular polysaccharides of
strains differ in repeating motifs and therefore vary in the number of hydroxyl groups, which, along with higher-order structure of the capsule, may contribute to the variation in hydrophobicity that we observed. We found that cell wall composition, in the context of chitin-chitosan content, does not influence CSH. For
, CSH correlated with phagocytosis by natural soil predator
Furthermore, capsular binding of the protective antibody (18B7), but not the nonprotective antibody (13F1), altered the CSH of
strains. Variability in CSH could be an important characteristic in comparing the biological properties of cryptococcal strains.
The interaction of a microbial cell with its environment is influenced by the biophysical properties of a cell. The affinity of the cell surface for water, defined by the cell surface hydrophobicity (CSH), is a biophysical parameter that varies among different strains of
The CSH influences the phagocytosis of the yeast by its natural predator in the soil, the amoeba. Studying variation in biophysical properties like CSH gives us insight into the dynamic host-predator interaction and host-pathogen interaction in a damage-response framework.
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited monogenic disorders, characterized by a progressive decline in kidney function due in part to the formation of ...fluid-filled cysts. While there is one FDA-approved therapy, it is associated with potential adverse effects, and all other clinical interventions are largely supportive. Insights into the cellular pathways underlying ADPKD have revealed striking similarities to cancer. Moreover, several drugs originally developed for cancer have shown to ameliorate cyst formation and disease progression in animal models of ADPKD. These observations prompted us to develop a high-throughput screening platform of cancer drugs in a quest to repurpose them for ADPKD. We screened ~8,000 compounds, including compounds with oncological annotations, as well as FDA-approved drugs, and identified 155 that reduced the viability of Pkd1-null mouse kidney cells with minimal effects on wild-type cells. We found that 109 of these compounds also reduced in vitro cyst growth of Pkd1-null cells cultured in a 3D matrix. Moreover, the result of the cyst assay identified therapeutically relevant compounds, including agents that interfere with tubulin dynamics and reduced cyst growth without affecting cell viability. Because it is known that several ADPKD therapies with promising outcomes in animal models failed to be translated to human disease, our platform also incorporated the evaluation of compounds in a panel of primary ADPKD and normal human kidney (NHK) epithelial cells. Although we observed differences in compound response amongst ADPKD and NHK cell preparation, we identified 18 compounds that preferentially affected the viability of most ADPKD cells with minimal effects on NHK cells. Our study identifies attractive candidates for future efficacy studies in advanced pre-clinical models of ADPKD.
Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been ...identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, we show herein the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay. We demonstrate the utility of this assay by generating dose-response curves on a comprehensive set of prior art inhibitors as well as hundreds of ALDH1A1 inhibitors synthesized in house. Finally, we established a screening paradigm using a pair of cell lines with low and high ALDH1A1 expression, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of over 1,000 small molecules.
Lengthy tuberculosis (TB) treatment is required to overcome the ability of a subpopulation of persistent
(
) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic ...remodeling, including induction of the Rel
-mediated stringent response. We developed a novel therapeutic DNA vaccine containing a fusion of the
gene with the gene encoding the immature dendritic cell-targeting chemokine, MIP-3α/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that intramuscular delivery of the
/
(fusion) vaccine or intranasal delivery of the
(non-fusion) vaccine potentiate isoniazid activity more than intramuscular delivery of the DNA vaccine expressing
alone in a chronic TB mouse model (absolute reduction of
burden: 0.63 log
and 0.5 log
colony-forming units, respectively; P=0.0002 and P=0.0052), inducing pronounced
-protective immune signatures. The combined approach involving intranasal delivery of the DNA
/
fusion vaccine demonstrated the greatest mycobactericidal activity together with isoniazid when compared to each approach alone (absolute reduction of
burden: 1.13 log
, when compared to the intramuscular vaccine targeting
alone; P<0.0001), as well as robust systemic and local Th1 and Th17 responses. This DNA vaccination strategy may be a promising adjunctive approach combined with standard therapy to shorten curative TB treatment, and also serves as proof of concept for treating other chronic bacterial infections.
The Mycobacterium avium complex (MAC) is one of the most prevalent causes of nontuberculous mycobacteria pulmonary infection in the United States, and yet it remains understudied. Current MAC ...treatment requires more than a year of intermittent to daily combination antibiotic therapy, depending on disease severity. In order to shorten and simplify curative regimens, it is important to identify the innate bacterial factors contributing to reduced antibiotic susceptibility, namely, antibiotic tolerance genes. In this study, we performed a genome-wide transposon screen to elucidate M. avium genes that play a role in the bacterium's tolerance to first- and second-line antibiotics. We identified a total of 193 unique M. avium mutants with significantly altered susceptibility to at least one of the four clinically used antibiotics we tested, including two mutants (in DFS55_00905 and DFS55_12730) with panhypersusceptibility. The products of the antibiotic tolerance genes we have identified may represent novel targets for future drug development studies aimed at shortening the duration of therapy for MAC infections.
The prolonged treatment required to eradicate Mycobacterium avium complex (MAC) infection is likely due to the presence of subpopulations of antibiotic-tolerant bacteria with reduced susceptibility to currently available drugs. However, little is known about the genes and pathways responsible for antibiotic tolerance in MAC. In this study, we performed a forward genetic screen to identify M. avium antibiotic tolerance genes, whose products may represent attractive targets for the development of novel adjunctive drugs capable of shortening the curative treatment for MAC infections.
(
), the causative agent of tuberculosis (TB), poses a global health challenge and is responsible for over a million deaths each year. Current treatment is lengthy and complex, and new, abbreviated ...regimens are urgently needed.
adapts to nutrient starvation, a condition experienced during host infection, by shifting its metabolism and becoming tolerant to the killing activity of bactericidal antibiotics. An improved understanding of the mechanisms mediating antibiotic tolerance in
can serve as the basis for developing more effective therapies. We performed a forward genetic screen to identify candidate
genes involved in tolerance to the two key first-line antibiotics, rifampin and isoniazid, under nutrient-rich and nutrient-starved conditions. In nutrient-rich conditions, we found 220 mutants with differential antibiotic susceptibility (218 in the rifampin screen and 2 in the isoniazid screen). Following
adaptation to nutrient starvation, 82 mutants showed differential antibiotic susceptibility (80 in the rifampin screen and 2 in the isoniazid screen). Using targeted mutagenesis, we validated the rifampin-hypersusceptible phenotype under nutrient starvation in
mutants lacking the following genes:
,
,
, and
. These findings shed light on potential therapeutic targets, which could help shorten the duration and complexity of antitubercular regimens.
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