Identification of Small-Molecule Inhibitors of the Salmonella FraB Deglycase Using a Live-Cell Assay.

Nontyphoidal salmonellosis is one of the most significant foodborne diseases in the United States and globally. There are no vaccines available for human use to prevent this disease, and only broad-spectrum antibiotics are available to treat complicated cases of the disease. However, antibiotic resistance is on the rise and new therapeutics are needed. We previously identified the Salmonella fraB gene, that mutation of causes attenuation of fitness in the murine gastrointestinal tract. The FraB gene product is encoded in an operon responsible for the uptake and utilization of fructose-asparagine (F-Asn), an Amadori product found in several human foods. Mutations in fraB cause an accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp), which is toxic to Salmonella. The F-Asn catabolic pathway is found only in the nontyphoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of Clostridium; it is not found in humans. Thus, targeting FraB with novel antimicrobials is expected to be Salmonella specific, leaving the normal microbiota largely intact and having no effect on the host. We performed high-throughput screening (HTS) to identify small-molecule inhibitors of FraB using growth-based assays comparing a wild-type Salmonella and a Δfra island mutant control. We screened 224,009 compounds in duplicate. After hit triage and validation, we found three compounds that inhibit Salmonella in an fra-dependent manner, with 50% inhibitory concentration (IC50) values ranging from 89 to 150 µM. Testing these compounds with recombinant FraB and synthetic 6-P-F-Asp confirmed that they are uncompetitive inhibitors of FraB with Ki' (inhibitor constant) values ranging from 26 to 116 µM. IMPORTANCE Nontyphoidal salmonellosis is a serious threat in the United States and globally. We have recently identified an enzyme, FraB, that when mutated renders Salmonella growth defective in vitro and unfit in mouse models of gastroenteritis. FraB is quite rare in bacteria and is not found in humans or other animals. Here, we have identified small-molecule inhibitors of FraB that inhibit the growth of Salmonella. These could provide the foundation for a therapeutic to reduce the duration and severity of Salmonella infections.

Use of tandem affinity-buffer exchange chromatography online with native mass spectrometry for optimizing overexpression and purification of recombinant proteins.

Purification of recombinant proteins typically entails overexpression in heterologous systems and subsequent chromatography-based isolation. While denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is routinely used to screen a variety of overexpression conditions (e.g., host, medium, inducer concentration, post-induction temperature and/or incubation time) and to assess the purity of the final product, its limitations, including aberrant protein migration due to compositional eccentricities or incomplete denaturation, often preclude firm conclusions regarding the extent of overexpression and/or purification. Therefore, we recently reported an automated liquid chromatography-mass spectrometry-based strategy that couples immobilized metal affinity chromatography (IMAC) with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS) to directly analyze cell lysates for the presence of target proteins. IMAC-OBE-nMS can be used to assess whether target proteins (1) are overexpressed in soluble form, (2) bind and elute from an IMAC resin, (3) oligomerize, and (4) have the expected mass. Here, we use four poly-His-tagged proteins to demonstrate the potential of IMAC-OBE-nMS for expedient optimization of overexpression and purification conditions for recombinant protein production.

The mycobacterial Rv1551 glycerol-3-phosphate acyltransferase enhances phospholipid biosynthesis in cell lysates of Escherichia coli.

Latent tuberculosis is caused by dormant Mycobacterium tuberculosis (Mtb) that is phenotypically tolerant to antibiotics. Dormant Mtb accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. The Rv1551 (PlsB1) gene is annotated as a putative glycerol-3-phosphate acyltransferase (GPAT) in the Mtb genome. GPAT catalyzes the first step of the glycerophospholipid biosynthetic pathway that synthesizes the lipid precursors for triacylglycerol biosynthesis. Although triacylglycerol biosynthesis is associated with Mtb dormancy, the functionality of the Rv1551 acyltransferase has not been investigated. We cloned the open reading frame of the Rv1551 acyltransferase and expressed it in Escherichia coli to study its function. We observed that E. coli cell lysates expressing Rv1551 displayed increased synthesis of phosphatidylglycerol, phosphatidylethanolamine and cardiolipin from radiolabeled glycerol-3-phosphate and fatty acyl-coenzyme A precursors. When cultured in medium supplemented with long-chain fatty acids, E. coli expressing Rv1551 exhibited significantly higher viable cell counts during the exponential and stationary phases. These results suggest that Rv1551 displays function as a GPAT by enhancing the synthesis of phospholipids from exogenously provided fatty acids in E. coli cell lysates. This is the first report showing that Rv1551 is a functional GPAT that catalyzes the initial step of glycerophospholipid biosynthesis in the mycobacterial cell.