A Global Approach to Identify Novel Broad-Spectrum Antibacterial Targets among Proteins of Unknown Function

Zalacaı́n, Magdalena; Biswas, Subhasis B.; Ingraham, Karen; Ambrad, Jennifer; Bryant, Alexander P.; Chalker, Alison F.; Iordănescu, S; Fan, Jing; Fan, Frank; Lunsford, R. Dwayne; O’Dwyer, Karen; Palmer, Leslie M.; So, Chi Y.; Sylvester, Daniel; Volker, Craig; Warren, Patrick V.; McDevitt, Damien; Brown, James R.; Holmes, David; Burnham, Martin K. R.

doi:10.1159/000076741

Cited by 76 publications

(67 citation statements)

References 56 publications

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“…S. pneumoniae uses the same enzymes to initiate phospholipid synthesis as S. aureus (Fig. 1A); however, the fakA gene in this organism (SP0443) is reported to be essential (22)(23)(24). The essential function in S. pneumoniae is unknown but is likely to be because of the regulatory properties of Fak rather than its role in phospholipid synthesis.…”

Section: Discussionmentioning

confidence: 98%

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

Parsons

Broussard

Bose

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

148

230

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Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus.T he pathway for the uptake and incorporation of host fatty acids (FA) by bacterial pathogens is important to understanding their physiology and determining if type II fatty acid synthesis (FASII) inhibitors will be useful antibacterial therapeutics. FASII is an energy-intensive process, and the advantage of being able to use host FA for membrane assembly obviously allows ATP to be diverted to the synthesis of other macromolecules. Gram-positive pathogens are capable of incorporating extracellular FA into their phospholipids. In species related to Streptococcus agalactiae, extracellular FA can completely replace endogenously synthesized FA, rendering the FASII inhibitors ineffective growth inhibitors if sufficient FA are present (1, 2). In contrast, FASII inhibitors exemplified by the enoyl-acyl carrier protein (ACP) reductase therapeutic AFN-1252 are effective against Staphylococcus aureus, even when extracellular FA are abundant (2, 3). A major impediment to our understanding of this diversity is that the pathway and enzymes responsible for the incorporation of extracellular FA is not established in the Firmicutes. A recent analysis of a ΔplsX knockout strain of S. aureus ruled out a role for either acyl-CoAs or acyl-ACP as intermediates in host FA metabolism and instead provided evidence for the existence of a new enzyme that phosphorylates FA, called FA kinase (Fak) (4) (Fig. 1A). Host FA are phosphorylated by Fak, and the acyl-PO 4 formed is either used by the PlsY glycerol-3-phosphate acyltransferase or converted to acyl-ACP by PlsX. The acyl-ACP may be either elongated by FASII or used by PlsC. Despite the strong evidence for their existence, the genes/proteins that encode this novel enzyme in FA metabolism were previously unidentif...

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Section: Discussionmentioning

confidence: 98%

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

Parsons

Broussard

Bose

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

148

230

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“…The systematic disruption of open reading frames has been undertaken either by transposon insertion tests or by plasmid insertion mutagenesis. This second strategy works best with naturally competent organisms, such as Streptococcus pneumoniae (69), and Bacillus subtilis (36). A similar approach based on insertion-duplication mutagenesis was recently described to screen for essential genes of Salmonella enterica serovar Typhimurium by using a bank of genomic fragments cloned into a conditionally replicating vector (35).…”

Section: Discussionmentioning

confidence: 99%

Identification of an Essential Gene ofListeria monocytogenesInvolved in Teichoic Acid Biogenesis

et al. 2006

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Listeria monocytogenes is a facultative intracellular gram-positive bacterium responsible for severe opportunistic infections in humans and animals. We had previously identified a gene encoding a putative UDP-Nacetylglucosamine 2-epimerase, a precursor of the teichoic acid linkage unit, in the genome of L monocytogenes strain EGD-e. This gene, now designated lmo2537, encodes a protein that shares 62% identity with the cognate epimerase MnaA of Bacillus subtilis and 55% identity with Cap5P of Staphylococcus aureus. Here, we addressed the role of lmo2537 in L. monocytogenes pathogenesis by constructing a conditional knockout mutant. The data presented here demonstrate that lmo2537 is an essential gene of L. monocytogenes that is involved in teichoic acid biogenesis. In vivo, the conditional mutant is very rapidly eliminated from the target organs of infected mice and thus is totally avirulent.

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“…The native gene was then replaced by the erythromycin resistance marker ermAM (27) by a precise allelic replacement method that minimizes polarity effects (42). Transformants were recovered on erythromycin and fucose, and genomic organization was confirmed by diagnostic PCR.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

et al. 2004

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Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, tRNA CAG Leu . Other heterologous nonsubstrate tRNA species, like tRNA GGT Thr , tRNA Phe , and tRNA TGC Ala , bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).Proteins that methylate tRNA at position G37 are ubiquitous in nature, and no cellular form has been found which does not possess this important activity. In bacteria, the enzyme tRNA (guanosine-1)-methyltransferase (1MGT or TrmD) modifies the subset of tRNAs that have a G at position 36 and recognize codons beginning with C, including those for leucine, proline, and arginine. It has been shown that methylation at G37 prevents frameshifting (7), and in strains of Salmonella enterica serovar Typhimurium lacking this activity, frameshifting occurs at selected proline codons (32,33). The deficiency of m 1 G in these strains also appears to have a more global effect on cell physiology as evidenced by changes in carbon source metabolism and sensitivity to amino acid analogs (24). In particular, lack of m 1 G is found to affect metabolism of thiamine and pantothenate (4, 6). TrmD activity has been shown to be important for growth of the gram-negative bacteria S. enterica serovar Typhimurium (5) and Escherichia coli (30), and recent studies suggest that TrmD is essential for growth of the grampositive organisms Streptococcus pneumoniae (36) and Bacillus subtilis (22).It has been shown that the entire tRNA structure is required for maximal catalytic activity of E. coli TrmD (18). For example, a derivative of tRNA CAG Leu with the D and T loops deleted is a very poor substrate for this enzyme. Subsequent studies have shown that the sequence G36pG37 is the key recognition element in tRNA (19) and that G37 must be in the correct position in space relative to important interactions elsewhere in the molecule (19). Enzymatic and chemical probing experiments have shown that the enzyme makes contacts in key sites in the anticodon loop (13).Although tRNA-modifying enzymes from E. coli have been well studied, little is known concerning modification enzymes from gram-positive bacteria. It is not clear, for instance, if gram-negative and gram-positive TrmD orthologs will display si...

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A Global Approach to Identify Novel Broad-Spectrum Antibacterial Targets among Proteins of Unknown Function

Cited by 76 publications

References 56 publications

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

Identification of an Essential Gene ofListeria monocytogenesInvolved in Teichoic Acid Biogenesis

Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

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