Rachel David

Bacterial physiology: Pass the (LPS) parcel

In Escherichia coli, lipopolysaccharide (LPS) is synthesized at the inner membrane and transported to the outer membrane for assembly. This is known to be mediated by LPS transport (Lpt) proteins: LptBFG forms a complex with the inner-membrane-bound LptC, which binds to LptA, and LptA interacts with LptD at the outer membrane, forming a trans-envelope bridge that connects the two membranes. Here, the authors elucidate the individual steps of LPS transport across the Lpt bridge. They found that LPS is extracted from the inner membrane by LptBFG, which then transports it to LptC; LPS is then transferred to LptA. Importantly, both transport events required ATP, so the authors propose that multiple rounds of ATP hydrolysis provide energy to push LPS from the inner to the outer membrane through the trans-envelope bridge.

Microbiome: Pathogens and commensals fight it out

Successful infection by enteric pathogens depends on the expression of pathogen virulence genes and the ability of the pathogens to outcompete the gut microbiota for nutrients.

Viral pathogenesis: Enabling HCV's need for fat.

hepatitis C virus (HCV) exploits host cell lipid metabolism pathways in infected hepatocytes, frequently resulting in pathological changes in lipid homeostasis. Liang and colleagues now identify an HCVactivated pathway involving inhibitor of NF-κB kinase subunit-α (IKKα) that modulates the host cell transcriptional programme for lipid metabolism genes and thus promotes viral particle production. Following on from previous work, the authors noted that depletion of IKKα (part of the IKK complex, which regulates the antiviral NF-κB signalling pathway) markedly impaired viral particle production and secretion, and specifically assembly. Viral particle assembly is known to involve association of the HCV core protein with host lipid droplets; the authors observed that lipid droplet numbers increased following HCV infection and that depletion of IKKα abrogated this effect. Notably, silencing other NF-κB pathway components had no effect on lipid droplet formation and HCV assembly, indicating that IKKαmediated induction of lipid droplet synthesis is NF-κB independent. So, how does HCV activate IKKα? First, the authors identified the HCV 3ʹ UTR as the component that triggers IKKα-dependent signalling and consequent lipid droplet production. The 3ʹ UTR was shown to be specifically recognized by the host DexDH/H helicase DDX3X, a putative pathogen recognition receptor, which redistributed in the cytoplasm and colocalized with the 3ʹ UTR and IKKα. Importantly, the interaction between IKKα and DDX3X resulted in IKKα phosphorylation (and therefore activation) and translocation to the nucleus. Sterol regulatory element-binding proteins (SREBPs) activate cholesterol and fatty acid synthesis and have been shown to be upregulated and activated in HCV-infected hepatocytes, so they might be involved in IKKα-mediated lipid droplet formation. Indeed, IKKα overexpression enhanced the expression of SREBPs and of their target genes, whereas IKKα silencing had the opposite effect. Moreover, and similarly to IKKα silencing, depletion of SREBP1 and SREBP2 impaired HCV particle production, probably owing to decreased lipid droplet formation. Thus, in the context of HCV infection, IKKα has a pro-viral role, facilitating viral particle assembly by promoting lipid droplet formation. Further work is now needed to determine whether chemical inhibitors of IKKα could be used as HCV antivirals. Rachel David V I R A L PAT H O G E N E S I S

Viral infection: Propelling vaccinia virus to the neighbours

cell, vaccinia virus downregulates signalling by the host RHO GTPase RHOA to weaken cortical actin, which acts as a physical barrier to exocytosis. Here, Way and colleagues identify the mechanism by which the virus inhibits RHOA signalling, revealing F11 as the first known viral protein to have a functional PDZ domain. PDZ domains are protein–protein interaction motifs that are involved in a range of cellular processes. Way and colleagues found that the highly conserved portion of F11 contains motifs that are characteristic of PDZ domains, and structural predictions suggested that it would adopt a PDZ-like fold. Interestingly, vaccinia virus with mutated F11 PDZ motifs formed fewer actin tails (which enhance viral spread), showed reduced cell–cell spread and released fewer infectious virions. These effects are indicative of failed RHOA inhibition, which suggests that the F11 PDZ domain regulates RHOA signalling. Like other GTPases, RHO proteins are regulated by RHO guanine exchange factors (RHOGEFs; which activate them) and RHO GTPaseactivating proteins (RHOGAPs; which inactivate them), both of which are known to have PDZ-binding motifs (PBMs). Through an in vitro pull-down assay, the authors identified two RHOGAPs, β-chimaerin and myosin IXa, that interacted with the F11 PDZ domain. Focusing on myosin IXa (which is known to regulate RHOA and cortical actin), they further observed that deletion of the myosin IXa PBM abolished its interaction with F11. Moreover, no virus-induced drop in active RHOA was observed in myosin IXa-depleted HeLa cells, which also had fewer actin tails and lower production of active virions than wild-type cells. Analysis of plaque formation in myosin IXa-depleted adherent cells also indicated less vaccinia virus cell–cell spread. By contrast, there was no significant difference between myosin IXadepleted cells and controls when cells were infected with F11-null vaccinia virus. These findings, together with the observation that fewer actin tails were formed in cells carrying GAP-mutant myosin IXa, suggest that myosin IXa GAP activity inhibits RHOA following interaction with the F11 PDZ domain. Importantly, depletion of myosin IXa did not affect the size of the plaques formed by a vaccinia virus carrying a mutant form of F11 that cannot bind RHOA, suggesting that myosin IXa acts as a GAP for RHOA only when RHOA is bound to F11. Consistent with this, the three proteins were shown to form complexes in infected cells. Thus, the authors conclude that F11 acts as a scaffold, using its PDZ domain to bring myosin IXa and RHOA together and ultimately promote vaccinia virus spread. This study identifies the first viral PDZ domain-containing protein; as F11 is present in other poxviruses, it is possible that this mechanism of promoting viral cell–cell spread is conserved. Rachel David V I R A L I N F E C T I O N

Host response: PF4 - platelets' poison.

Platelet factor 4 is the effector molecule used by platelets to kill P. falciparum in infected erythrocytes.

Microbiome: A bacterial trigger for liver cancer

Plasmodium falciparum has 60 var genes encoding distinct antigenic forms of the virulence protein PfEMP1 (P. falciparum erythrocyte membrane protein 1). The parasite expresses only one var gene at any time point during infection to avoid detection by the immune system, but the mechanism controlling the silencing of the other 59 var genes was unknown. Now, Jiang et al. show that var gene silencing is regulated by the histone lysine methyltransferase PfSETvs (previously known as PfSET2). Knockout of PfSETvs led to a strong reduction in trimethylation of histone H3 lysine 36 along the entire body of var genes and to transcription of almost all var gene family members. Notably, confocal microscopy showed that knockout of PfSETvs resulted in the expression of multiple PfEMP1 variants at the surface of infected red blood cells.

Techniques and applications: Finding out how antimicrobials work

Identifying the mechanism of action of antimicrobials remains a challenge and is a major limitation for the drug discovery process. Here, Pogliano and colleagues test the ability of bacterial cytological profiling (BCP) to determine the mechanism of action of antimicrobials. BCP involves the treatment of bacterial cells with a compound and the subsequent analysis of morphological changes to identify the cellular pathway targeted. Using this technique, the authors could distinguish between inhibitory compounds that target different cellular pathways (for example, protein synthesis or DNA replication), as they generated distinct cytological profiles. Moreover, they used BCP to show that the antibacterial compound spirohexenolide A probably kills bacterial cells by disrupting the cytoplasmic membrane. Thus, BCP could be used to rapidly characterize the mechanism of action of antimicrobials and, in combination with other approaches, to determine the precise target in the pathway.

Bacterial evolution: the road to E. faecium pathogenesis.

A new study offers insights into the changes that drove the switch from a commensal to a pathogenic lifestyle in Enterococcus faecium.

Antimicrobials: Why zinc is bad for bacteria

Zn is known to have antibacterial properties, although the mechanism by which it acts was unknown. Here, the authors have found that ZnII competes with MnII for binding to the Streptococcus pneumoniae protein PsaA, a solute-binding protein that transports MnII into the cell to manage oxidative stress, among other functions. Although ZnII showed lower affinity for PsaA than MnII, the complex formed by ZnII and PsaA was more thermally stable. Furthermore, increasing the ZnII/MnII ratio, which would decrease MnII uptake, led to inhibition of S. pneumoniae growth in vitro owing to increased susceptibility to oxidative stress, as well as increased susceptibility to killing by polymorphonuclear leukocytes. Consistent with this, mice infected with S. pneumoniae showed a significant increase in ZnII levels compared with controls in tissue samples collected 48 hours post-infection. So, it seems that ZnII is toxic to S. pneumoniae because it inhibits MnII uptake.

Bacterial pathogenesis: A competitive edge for Salmonella.

S. Typhimurium is resistant to the effects of calprotectin owing to its Zn transporter and thus has a competitive growth advantage in the inflamed host gut.