Yu-Feng Yao

Hcp family proteins secreted via the type VI secretion system coordinately regulate Escherichia coli K1 interaction with human brain microvascular endothelial cells.

ABSTRACT Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. Based on sequence analysis, we found that a cluster of E scherichia coli virulence factors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli-HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.

HbiF regulates type 1 fimbriation independently of FimB and FimE

ABSTRACT Type 1 fimbriae have been suggested to play a role in the pathogenesis of extraintestinal Escherichia coli infection. Type 1 fimbriation in E. coli is phase variable and known to be dependent upon FimB and FimE, the two recombinases that invert the molecular switch fimS and control the expression of the downstream fim operon. Here we showed that HbiF, a novel site-specific recombinase, inverted fimS independently of FimB and FimE. HbiF-mediated fimS inversion appeared to be predominantly switching from “off” (termed OFF) to “on” (termed ON) orientation. This is different from the fimS inversion mediated by either FimB (bidirectional ON to OFF and OFF to ON) or FimE (unidirectional ON to OFF). Constitutive expression of the hbiF gene in E. coli resulted in a fimS-locked-ON phenotype, which resulted in the pathogenic E. coli K1 strain being incapable of inducing a high degree of bacteremia in neonatal rats. Discovery of HbiF-mediated OFF-to-ON fimS switching provides a new opportunity to develop a strategy for the prevention and therapy of extraintestinal E. coli infection including bacteremia and meningitis.

Flagella Promote Escherichia coli K1 Association with and Invasion of Human Brain Microvascular Endothelial Cells

ABSTRACT Escherichia coli containing the K1 capsule is the leading cause of gram-negative meningitis, but the pathogenesis of this disease is not completely understood. Recent microarray experiments in which we compared the gene expression profile of E. coli K1 associated with human brain microvascular endothelial cells (HBMEC) to the gene expression profile of E. coli K1 not associated with HBMEC revealed that there was a threefold increase in the expression of the fliI gene, encoding an ATP synthase involved in flagellar synthesis and motility, in HBMEC-associated E. coli. In this study, we examined the role of flagella in E. coli K1 association with and invasion of HBMEC by constructing isogenic ΔflhDC, ΔfliI, ΔfliC, and ΔcheW mutants that represented each class of flagellar genes. Mutations that affected the flagellum structure and flagellum formation (ΔflhDC, ΔfliI, and ΔfliC) resulted in significant defects in motility, as well as in HBMEC association and invasion, compared to the characteristics of the wild-type strain when preparations were examined with or without centrifugation. Transcomplementation with the corresponding genes restored the levels of these mutants to the levels of the parent strain. These findings suggest that the HBMEC association and invasion defects of the mutants are most likely related to flagella and less likely due to their motility defects. This conclusion was supported by our demonstration that the cheW mutant was not motile but was able to associate with and invade HBMEC. In addition, purified recombinant flagellin reduced the association of the wild-type strain with HBMEC by ∼40%, while it had no effect on the fliC mutants association with HBMEC. Together, these findings indicate that flagella promote E. coli K1 binding to HBMEC.

Genomic Comparison of Escherichia coli K1 Strains Isolated from the Cerebrospinal Fluid of Patients with Meningitis

ABSTRACT Escherichia coli is a major cause of enteric/diarrheal diseases, urinary tract infections, and sepsis. E. coli K1 is the leading gram-negative organism causing neonatal meningitis, but the microbial basis of E. coli K1 meningitis is incompletely understood. Here we employed comparative genomic hybridization to investigate 11 strains of E. coli K1 isolated from the cerebrospinal fluid (CSF) of patients with meningitis. These 11 strains cover the majority of common O serotypes in E. coli K1 isolates from CSF. Our data demonstrated that these 11 strains of E. coli K1 can be categorized into two groups based on their profile for putative virulence factors, lipoproteins, proteases, and outer membrane proteins. Of interest, we showed that some open reading frames (ORFs) encoding the type III secretion system apparatus were found in group 2 strains but not in group 1 strains, while ORFs encoding the general secretory pathway are predominant in group 1 strains. These findings suggest that E. coli K1 strains isolated from CSF can be divided into two groups and these two groups of E. coli K1 may utilize different mechanisms to induce meningitis.

Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence.

The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix–turn–helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms.

Reversible lysine acetylation is involved in DNA replication initiation by regulating activities of initiator DnaA in Escherichia coli.

The regulation of chromosomal replication is critical and the activation of DnaA by ATP binding is a key step in replication initiation. However, it remains unclear whether and how the process of ATP-binding to DnaA is regulated. Here, we show that DnaA can be acetylated, and its acetylation level varies with cell growth and correlates with DNA replication initiation frequencies in E. coli. Specifically, the conserved K178 in Walker A motif of DnaA can be acetylated and its acetylation level reaches the summit at the stationary phase, which prevents DnaA from binding to ATP or oriC and leads to inhibition of DNA replication initiation. The deacetylation process of DnaA is catalyzed by deacetylase CobB. The acetylation process of DnaA is mediated by acetyltransferase YfiQ, and nonenzymatically by acetyl-phosphate. These findings suggest that the reversible acetylation of DnaA ensures cells to respond promptly to environmental changes. Since Walker A motif is universally distributed across organisms, acetylation of Walker A motif may present a novel regulatory mechanism conserved from bacteria to eukaryotes.

Acetylation Regulates Survival of Salmonella enterica Serovar Typhimurium under Acid Stress

ABSTRACT The ability to acetylate lysine residues is conserved across organisms, and acetylation of lysine residues plays important roles in various cellular functions. Maintaining intracellular pH homeostasis is crucial for the survival of enteric bacteria in the acidic gastric tract. It has been shown that eukaryotes can stabilize the intracellular pH by histone deacetylation. However, it remains unknown whether bacteria can utilize a reversible protein acetylation system to adapt to an acidic environment. Here we demonstrate that protein acetylation/deacetylation is critical for Salmonella enterica serovar Typhimurium to survive in an acidic environment. We used RNA sequencing to analyze the transcriptome patterns under acid stress and found that the transcriptional levels of genes involved in NAD+/NADH metabolism were significantly changed, leading to an increase in the intracellular NAD+/NADH ratio. Moreover, acid stress downregulated the transcriptional level of pat, encoding acetyltransferase, and genes cyaA and crp, encoding adenylate cyclase and cyclic AMP receptor protein, respectively, which are positive regulators of pat. It was found that the acid signal alerts the tricarboxylic acid cycle to promote the consumption of acetyl coenzyme A (Ac-CoA), an acetyl group donor for the acetylation reaction. A lowered acetylation level not only was the bacterial response to acid stress but also increased the survival rate of S. Typhimurium under acid stress. The pat deletion mutant had a more stable intracellular pH, which paralleled the higher survival rate after acid treatment compared with that of both the wild-type strain and the cobB (encoding deacetylase) deletion mutant. Our data indicate that bacteria can downregulate the protein acetylation level to prevent the intracellular pH from further falling under acid stress, and this work may provide a new perspective to understand the bacterial acid resistance mechanism.

NlpI facilitates deposition of C4bp on Escherichia coli by blocking classical complement-mediated killing, which results in high-level bacteremia.

ABSTRACT Neonatal meningitis Escherichia coli (NMEC) is the most common Gram-negative organism that is associated with neonatal meningitis, which usually develops as a result of hematogenous spread of the bacteria. There are two key pathogenesis processes for NMEC to penetrate into the brain, the essential step for the development of E. coli meningitis: a high-level bacteremia and traversal of the blood-brain barrier (BBB). Our previous study has shown that the bacterial outer membrane protein NlpI contributes to NMEC binding to and invasion of brain microvascular endothelial cells, the major component cells of the BBB, suggesting a role for NlpI in NMEC crossing of the BBB. In this study, we showed that NlpI is involved in inducing a high level of bacteremia. In addition, NlpI contributed to the recruitment of the complement regulator C4bp to the surface of NMEC to evade serum killing, which is mediated by the classical complement pathway. NlpI may be involved in the interaction between C4bp and OmpA, which is an outer membrane protein that directly interacts with C4bp on the bacterial surface. The involvement of NlpI in two key pathogenesis processes of NMEC meningitis may make this bacterial factor a potential target for prevention and therapy of E. coli meningitis.

Protein Acetylation and Its Role in Bacterial Virulence

Protein acetylation is a universal post-translational modification which is found in both eukaryotes and prokaryotes. This process is achieved enzymatically by the protein acetyltransferase Pat, and nonenzymatically by metabolic intermediates (e.g., acetyl phosphate) in bacteria. Protein acetylation plays a role in bacterial chemotaxis, metabolism, DNA replication, and other cellular processes. Recently, accumulating evidence has suggested that protein acetylation might be involved in bacterial virulence because a number of bacterial virulence factors are acetylated. In this review, we summarize the progress in understanding bacterial protein acetylation and discuss how it mediates bacterial virulence.

The Bacterial Two-Hybrid System Uncovers the Involvement of Acetylation in Regulating of Lrp Activity in Salmonella Typhimurium

N𝜀-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat – or deacetylase CobB-mediated acetylation. Then, the in vitro (de)acetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36) in helix-turn-helix (HTH) DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes.