Acyl-AcpB, a FabT corepressor in Streptococcus pyogenes.

Membranes are a universal barrier to all cells. Phospholipids, essential bacterial membrane components, are composed of a polar head and apolar fatty acid (FA) chains. Most bacterial FAs are synthesized by the Type II FA synthesis pathway (FASII). In Streptococcaceae, Enterococci, and Lactococcus lactis, a unique feedback mechanism controls the FASII gene expression. FabT, encoded in the FASII main locus, is the repressor, and it is activated by long-chain acyl-acyl carrier protein (acyl-ACP). Many Streptococci, Enterococcus faecalis, but not L. lactis, possess two ACPs. The AcpA-encoding gene is within the FASII locus and is coregulated with the FASII genes. Acyl-AcpA is the end product of FASII. The AcpB-encoding gene is in operon with plsX encoding an acyl-ACP:phosphate acyltransferase. The role of acyl-AcpB as FabT corepressor is controversial. Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possesses AcpB. In this study, by comparing the expression of FabT-controlled genes in an acpB-deleted mutant with those in a wild-type and in a fabT mutant strain, grown in the presence or absence of exogenous FAs, we show that AcpB is the S. pyogenes FabT main corepressor. Its deletion impacts membrane FA composition and bacterial adhesion to eucaryotic cells, highlighting the importance of FASII control. Importance Membrane composition is crucial for bacterial growth or interaction with the environment. Bacteria synthesize fatty acids (FAs), membrane major constituents, via the Type II FAS (FASII) pathway. Streptococci control the expression of the FASII genes via a transcriptional repressor, FabT, with acyl-acyl carrier proteins (ACPs) as corepressor. Streptococcus pyogenes that causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections possesses two ACPs. acpA, but not acpB, is a FASII gene. In this study, we show that acyl-AcpBs are FabT main corepressors. Also, AcpB deletion has consequences on the membrane FA composition and bacterial adhesion to host cells. In addition to highlighting the importance of FASII control in the presence of exogeneous FAs for the adaptation of bacteria to their environment, our data indicate that FASII gene repression is mediated by a corepressor whose gene expression is not repressed in the presence of exogenous FAs.

A Novel CovS Variant Harbored by a Colonization Strain Reduces Streptococcus pyogenes Virulence.

Streptococcus pyogenes, also known as group A Streptococcus, causes a wide variety of diseases ranging from mild noninvasive to severe invasive infections. To identify possible causes of colonization-to-invasive switches, we determined the genomic sequences of 10 isolates from five pairs each composed of an invasive strain and a carriage strain originating from five infectious clusters. Among them, one pair displayed a single-nucleotide difference in covS, encoding the sensor histidine kinase of the two-component CovRS system that controls the expression of 15% of the genome. In contrast to previously described cases where the invasive strains harbor nonfunctional CovS proteins, the carriage strain possessed the mutation covST115C, leading to the replacement of the tyrosine at position 39 by a histidine. The CovSY39H mutation affected the expression of the genes from the CovR regulon in a unique fashion. Genes usually overexpressed in covS mutant strains were underexpressed and vice versa. Furthermore, the covS mutant strain barely responded to the addition of the CovS-signaling compounds Mg2+ and LL-37. The variations in the accumulation of two virulence factors paralleled the transcription modifications. In addition, the covST115C mutant strain showed less survival than its wild-type counterpart in murine macrophages. Finally, in two murine models of infection, the covS mutant strain was less virulent than the wild-type strain. Our study suggests that the CovSY39H protein compromises CovS phosphatase activity and that this yields a noninvasive strain. IMPORTANCE Streptococcus pyogenes, also known as group A Streptococcus, causes a wide variety of diseases, leading to 517,000 deaths yearly. The two-component CovRS system, which responds to MgCl2 and the antimicrobial peptide LL-37, controls the expression of 15% of the genome. Invasive strains may harbor nonfunctional CovS sensor proteins that lead to the derepression of most virulence genes. We isolated a colonization strain that harbors a novel covS mutation. This mutant strain harbored a transcriptome profile opposite that of other covS mutant strains, barely responded to environmental signals, and was less virulent than the wild-type strain. This supports the importance of the derepression of the expression of most virulence genes, via mutations that impact the phosphorylation of the regulator CovR, for favoring S. pyogenes invasive infections.

Type II Fatty Acid Synthesis Pathway and Cyclopropane Ring Formation Are Dispensable during Enterococcus faecalis Systemic Infection.

Enterococcus faecalis, a multiple antibiotic-resistant Gram-positive bacterium, has emerged as a serious nosocomial pathogen. Here, we used a genetic approach to characterize the strategies used by E. faecalis to fulfill its requirements for endogenous fatty acid (FA) synthesis in vitro and in vivo. The type II fatty acid synthesis (FASII) pathway is encoded by two operons and two monocistronic genes. Expression of all of these genes is repressed by exogenous FAs, which are incorporated into the E. faecalis membrane and modify its composition. Deletion of nine genes of the 12-gene operon abolished growth in an FA-free medium. Addition of serum, which is lipid rich, restored growth. Interestingly, the E. faecalis membrane contains cyclic fatty acids that modify membrane properties but that are unavailable in host serum. The cfa gene that encodes the cyclopropanation process is located in a locus independent of the FASII genes. Its deletion did not alter growth under the conditions tested, but yielded bacteria devoid of cyclic FAs. No differences were observed between mice infected with wild-type (WT) or with FASII or cyclopropanation mutant strains, in terms of bacterial loads in blood, liver, spleen, or kidneys. We conclude that in E. faecalis, neither FASII nor cyclopropanation enzymes are suitable antibiotic targets. IMPORTANCE Membrane lipid homeostasis is crucial for bacterial physiology, adaptation, and virulence. Fatty acids are constituents of the phospholipids that are essential membrane components. Most bacteria incorporate exogenous fatty acids into their membranes. Enterococcus faecalis has emerged as a serious nosocomial pathogen that is responsible for urinary tract infections, bacteremia, and endocarditis and is intrinsically resistant to numerous antibiotics. E. faecalis synthesizes saturated and unsaturated fatty acids, as well as cyclic fatty acids that are not found in the human host. Here, we characterized mutant strains deficient in fatty acid synthesis and modification using genetic, biochemical, and in vivo approaches. We conclude that neither the fatty acid synthesis pathway nor the cyclopropanation enzyme are suitable targets for E. faecalis antibiotic development.

Oxidative stress is intrinsic to staphylococcal adaptation to fatty acid synthesis antibiotics.

Antibiotics inhibiting the fatty acid synthesis pathway (FASII) of the major pathogen Staphylococcus aureus reach their enzyme targets, but bacteria continue growth by using environmental fatty acids (eFAs) to produce phospholipids. We assessed the consequences and effectors of FASII-antibiotic (anti-FASII) adaptation. Anti-FASII induced lasting expression changes without genomic rearrangements. Several identified regulators affected the timing of adaptation outgrowth. Adaptation resulted in decreased expression of major virulence factors. Conversely, stress responses were globally increased and adapted bacteria were more resistant to peroxide killing. Importantly, pre-exposure to peroxide led to faster anti-FASII-adaptation by stimulating eFA incorporation. This adaptation differs from reports of peroxide-stimulated antibiotic efflux, which leads to tolerance. In vivo, anti-FASII-adapted S. aureus killed the insect host more slowly but continued multiplying. We conclude that staphylococcal adaptation to FASII antibiotics involves reprogramming, which decreases virulence and increases stress resistance. Peroxide, produced by the host to combat infection, favors anti-FASII adaptation.

A Streptococcus pyogenes DegV protein regulates the membrane lipid content and limits the formation of extracellular vesicles.

Membranes contain lipids that are composed of fatty acids (FA) and a polar head. Membrane homeostasis is crucial for optimal bacterial growth and interaction with the environment. Bacteria synthesize their FAs via the FASII pathway. Gram-positive bacteria can incorporate exogenous FAs which need to be phosphorylated to become substrate of the lipid biosynthetic pathway. In many species including staphylococci, streptococci and enterococci, this phosphorylation is carried out by the Fak complex, which is composed of two subunits, FakA and FakB. FakA is the kinase. FakB proteins are members of the DegV family, proteins known to bind FAs. Two or three FakB types have been identified depending on the bacterial species and characterized by their affinity for saturated and/or unsaturated FAs. Some species such as Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possess an uncharacterized additional DegV protein. We identify here this DegV member as a fourth FakB protein, named FakB4. The fakB4 gene is co-regulated with FASII genes suggesting an interaction with endogenous fatty acids. fakB4 deletion has no impact on membrane phospholipid composition nor on the percentage of other major lipids. However, the fakB4 mutant strain produced more lipids and more extracellular membrane vesicles than the wild-type strain. This suggests that FakB4 is involved in endogenous FA binding and controls FA storage or catabolism resulting in a limitation of extracellular FA release via membrane vesicles.

FabT, a Bacterial Transcriptional Repressor That Limits Futile Fatty Acid Biosynthesis.

Phospholipids are vital membrane constituents that determine cell functions and interactions with the environment. For bacterial pathogens, rapid adjustment of phospholipid composition to changing conditions during infection can be crucial for growth and survival. Fatty acid synthesis (FASII) regulators are central to this process. This review puts the spotlight on FabT, a MarR-family regulator of FASII characterized in streptococci, enterococci, and lactococci. Roles of FabT in virulence, as reported in mouse and nonhuman primate infection models, will be discussed. We present FabT structure, the FabT regulon, and changes in FabT regulation according to growth conditions. A unique feature of FabT concerns its modulation by an unconventional corepressor, acyl-acyl-carrier protein (ACP). Some bacteria express two ACP proteins, which are distinguished by their interactions with endogenous or exogenous fatty acid sources, one of which causes strong FabT repression. This system seems to allow preferred use of environmental fatty acids, thereby saving energy by limiting futile FASII activity. Control of fabT expression and FabT activity link various metabolic pathways to FASII. The various physiological consequences of FabT loss summarized here suggest that FabT has potential as a narrow range therapeutic target.

Streptococcus pyogenes infects human endometrium by limiting the innate immune response.

Group A Streptococcus (GAS), a Gram-positive human-specific pathogen, yields 517,000 deaths annually worldwide, including 163,000 due to invasive infections and among them puerperal fever. Before efficient prophylactic measures were introduced, the mortality rate for mothers during childbirth was approximately 10%; puerperal fever still accounts for over 75,000 maternal deaths annually. Yet, little is known regarding the factors and mechanisms of GAS invasion and establishment in postpartum infection. We characterized the early steps of infection in an ex vivo infection model of the human decidua, the puerperal fever portal of entry. Coordinate analysis of GAS behavior and the immune response led us to demonstrate that (a) GAS growth was stimulated by tissue products; (b) GAS invaded tissue and killed approximately 50% of host cells within 2 hours, and these processes required SpeB protease and streptolysin O (SLO) activities, respectively; and (c) GAS impaired the tissue immune response. Immune impairment occurred both at the RNA level, with only partial induction of the innate immune response, and protein level, in an SLO- and SpeB-dependent manner. Our study indicates that efficient GAS invasion of the decidua and the restricted host immune response favored its propensity to develop rapid invasive infections in a gynecological-obstetrical context.