Luís Jaime Mota

The bacterial injection kit: Type III secretion systems

Type III secretion (T3S) systems are widespread among Gram‐negative bacteria pathogenic for animals and plants, including Yersinia spp., Salmonella spp., Shigella spp., enteropathogenic Escherichia coli, enterohaemorrhagic E. coli, or Pseudomonas spp. T3S systems allow bacteria to inject virulence proteins, called T3S effectors, into the cytosol of their eukaryotic host cells. These virulence factors will paralyze or reprogram the eukaryotic cell to the benefit of the pathogen. T3S effectors display a large repertoire of biochemical activities and modulate the function of crucial host regulatory molecules such as small guanosine triphosphate (GTP)‐binding proteins, mitogen‐activated protein kinases (MAPKs), nuclear factor (NF)‐κB, or phosphoinositides. The activity of T3S effectors allows bacteria, for example, to invade non‐phagocytic cells or to inhibit phagocytosis, to downregulate or promote pro‐inflammatory responses, to induce apoptosis, to prevent autophagy, or to modulate intracellular trafficking. In this review, we present the most recent advances in the understanding of the mode of action of T3S effectors. We highlight the biochemical activities of these eukaryotic‐like bacterial proteins that are shared among pathogens carrying T3S systems and the sequence, structural and functional resemblances between T3S effectors and eukaryotic proteins.

Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica

The length of the needle ending the Yersinia Ysc injectisome is determined by YscP, a protein acting as a molecular ruler. In addition, YscP is required for Yop secretion. In the present paper, by a systematic deletion analysis, we localized accurately the region required for Yop secretion between residues 405 and 500. As this C‐terminal region of YscP has also been shown to control needle length it probably represents the substrate specificity switch of the machinery. By a bioinformatics analysis, we show that this region has a globular structure, an original α/β fold, a P‐x‐L‐G signature and presumably no catalytic activity. In spite of very limited sequence similarities, this structure is conserved among the proteins that are presumed to control the needle length in many different injectisomes and also among members of the FliK family, which control the flagellar hook length. This region thus represents a new protein domain that we called T3S4 for Type III secretion substrate specificity switch. The T3S4 domain of YscP can be replaced by the T3S4 domain of AscP (Aeromonas salmonicida) or PscP (Pseudomonas aeruginosa) but not by the one from FliK, indicating that in spite of a common global structure, these domains need to fit their partner proteins in the secretion apparatus.

Salmonella Inhibits Retrograde Trafficking of Mannose-6-Phosphate Receptors and Lysosome Function

Subversive Salmonella Salmonella is one of the most intensively studied bacterial pathogens. The Salmonella-containing vacuole (SCV) has a paradoxical lysosome-like composition: On one hand, the SCV membrane is highly enriched in lysosomal membrane glycoproteins and SCVs are accessible to endolysosomal content, but on the other hand, SCV lumen is relatively devoid of lysosomal hydrolytic enzymes that are delivered by the mannose-6-phosphate receptor pathway. McGourty et al. (p. 963) resolve this paradox by showing that the Salmonella effector SifA interferes with the trafficking of mannose-6-phosphate receptors. This causes misrouting and secretion of lysosomal enzymes and reduces the hydrolytic activity of lysosomes. Intracellular growth of Salmonella was reduced in cells with enhanced lysosomal enzyme activity. A bacterial pathogen interferes with intracellular trafficking of receptors needed for host cell lysosomal-enzyme targeting. Salmonella enterica is an intracellular bacterial pathogen that replicates within membrane-bound vacuoles through the action of effector proteins translocated into host cells. Salmonella vacuoles have characteristics of lysosomes but are reduced in hydrolytic enzymes transported by mannose-6-phosphate receptors (MPRs). We found that the effector SifA subverted Rab9-dependent retrograde trafficking of MPRs, thereby attenuating lysosome function. This required binding of SifA to its host cell target SKIP/PLEKHM2. Furthermore, SKIP regulated retrograde trafficking of MPRs in noninfected cells. Translocated SifA formed a stable complex with SKIP and Rab9 in infected cells. Sequestration of Rab9 by SifA-SKIP accounted for the effect of SifA on MPR transport and lysosome function. Growth of Salmonella increased in cells with reduced lysosomal activity and decreased in cells with higher lysosomal activity. These results suggest that Salmonella vacuoles undergo fusion with lysosomes whose potency has been reduced by SifA.

The discovery of SycO highlights a new function for type III secretion effector chaperones

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone‐binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation‐prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.

The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles

Intracellular replication of Salmonella enterica occurs in membrane‐bound compartments, called Salmonella‐containing vacuoles (SCVs). Following invasion of epithelial cells, most SCVs migrate to a perinuclear region and replicate in close association with the Golgi network. The association of SCVs with the Golgi is dependent on the Salmonella‐pathogenicity island‐2 (SPI‐2) type III secretion system (T3SS) effectors SseG, SseF and SifA. However, little is known about the dynamics of SCV movement. Here, we show that in epithelial cells, 2 h were required for migration of the majority of SCVs to within 5 μm from the microtubule organizing centre (MTOC), which is located in the same subcellular region as the Golgi network. This initial SCV migration was saltatory, bidirectional and microtubule‐dependent. An intact Golgi, SseG and SPI‐2 T3SS were dispensable for SCV migration to the MTOC, but were essential for maintenance of SCVs in that region. Live‐cell imaging between 4 and 8 h post invasion revealed that the majority of wild‐type SCVs displaced less than 2 μm in 20 min from their initial starting positions. In contrast, between 6 and 8 h post invasion the majority of vacuoles containing sseG, sseF or ssaV mutant bacteria displaced more than 2 μm in 20 min from their initial starting positions, with some undergoing large and dramatic movements. Further analysis of the movement of SCVs revealed that large displacements were a result of increased SCV speed rather than a change in their directionality, and that SseG influences SCV motility by restricting vacuole speed within the MTOC/Golgi region. SseG might function by tethering SCVs to Golgi‐associated molecules, or by controlling microtubule motors, for example by inhibiting kinesin recruitment or promoting dynein recruitment.

Salmonella-induced tubular networks

Salmonella virulence relies on its capacity to replicate inside various cell types in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). A unique feature of Salmonella-infected cells is the presence of tubular structures originating from and connected to the SCV, which often extend throughout the cell cytoplasm. These tubules include the well-studied Salmonella-induced filaments (SIFs), enriched in lysosomal membrane proteins. However, recent studies revealed that the Salmonella-induced tubular network is more extensive than previously thought and includes three types of tubules distinct from SIFs: sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 (SCAMP3) tubules and lysosome-associated membrane protein 1 (LAMP1)-negative tubules. In this review, we examine the molecular mechanisms involved in the formation of Salmonella-induced tubular networks and discuss the importance of the tubules for Salmonella virulence and establishment of a Salmonella intracellular replicative niche.

SCAMP3 is a component of the Salmonella-induced tubular network and reveals an interaction between bacterial effectors and post-Golgi trafficking

Salmonella enterica are facultative intracellular bacterial pathogens that proliferate within host cells in a membrane‐bounded compartment, the Salmonella‐containing vacuole (SCV). Intracellular replication of Salmonella is mediated by bacterial effectors translocated on to the cytoplasmic face of the SCV membrane by a type III secretion system. Some of these effectors manipulate the host endocytic pathway, resulting in the formation in epithelial cells of tubules enriched in late endosomal markers, known as Salmonella‐induced filaments (SIFs). However, much less is known about possible interference of Salmonella with the secretory pathway. Here, a small‐interference RNA screen revealed that secretory carrier membrane proteins (SCAMPs) 2 and 3 contribute to the maintenance of SCVs in the Golgi region of HeLa cells. This is likely to reflect a function of SCAMPs in vacuolar membrane dynamics. Moreover, SCAMP3, which accumulates on the trans‐Golgi network in uninfected cells, marked tubules induced by Salmonella effectors that overlapped with SIFs but which also comprised distinct tubules lacking late endosomal proteins. We propose that SCAMP3 tubules reflect a manipulation of specific post‐Golgi trafficking that might allow Salmonella to acquire nutrients and membrane, or to control host immune responses.

Mode of action of AraR, the key regulator of l-arabinose metabolism in Bacillus subtilis

The AraR protein is a negative regulator involved in l‐arabinose‐inducible expression of the Bacillus subtilis araABDLMNPQ‐abfA metabolic operon and of the araE/araR genes that are organized as a divergent transcriptional unit. The two ara gene clusters are found at different positions in the bacterial chromosome. AraR was overproduced in Escherichia coli and purified to more than 95% homogeneity. AraR binds specifically to DNA fragments carrying the promoter region of the ara genes. DNase I protection assays showed that AraR binds to two sequences within the promoters of the araABDLMNPQ‐abfA operon and the araE gene, and to one sequence in the araR promoter. The AraR target sequences are palindromic and share high identity, defining a 16 bp AraR consensus operator sequence showing half‐symmetry, ATTTGTAC. Binding of AraR to DNA was inhibited by l‐arabinose but not by other sugars. The two operator sites within the araABDLMNPQ‐abfA operon and araE promoters are located on the same side of the DNA helix, and a pattern of enhanced and diminished DNase I cleavage was observed between them, but not in the araR promoter. Quantitative DNase I footprinting in DNA templates containing one, two or three AraR binding sites showed that the repressor binds cooperatively to the two operator sites within the metabolic operon and araE promoters but not to the site located in the araR promoter. These results are consistent with two modes for AraR transcriptional repression that might correlate with different physiological requirements: a high level of repression is achieved by DNA bending requiring two in‐phase operator sequences (metabolic operon and araE transport gene), whereas binding to a single operator, which autoregulates araR expression, is 10‐fold less effective.

Effect of low- and high-virulence Yersinia enterocolitica strains on the inflammatory response of human umbilical vein endothelial cells.

ABSTRACT Pathogenic strains of Yersinia spp. inject a set of Yop effector proteins into eukaryotic cells by using a plasmid-encoded type III secretion system. In this study, we analyzed the inflammatory response of human umbilical vein endothelial cells (HUVECs) after infection with different Yersinia enterocolitica strains. We found that both expression of intercellular adhesion molecule 1 and release of the cytokines interleukin-6 (IL-6) and IL-8 by HUVECs are downregulated in a YopP-dependent way, demonstrating that YopP plays a major role in the inflammatory response of these cells. Infection of HUVECs with several low-virulence (biotype 2, 3, and 4) and high-virulence (biotype 1B) Y. enterocolitica strains showed that biotype 1B isolates are more efficient in inhibiting the inflammatory response than low-virulence Y. enterocolitica strains and that this effect depends on the time of contact. We extended the results of Ruckdeschel et al. and found that on the basis of the presence or absence of arginine-143 of YopP (K. Ruckdeschel, K. Richter, O. Mannel, and J. Heesemann, Infect. Immun. 69:7652-7662, 2001) all the Y. enterocolitica strains used fell into two groups, which correlate with the low- and high-virulence phenotypes. In addition, we found that high-virulence strains inject more YopP into the cytosol of eukaryotic target cells than do low-virulence strains.

Control of the Arabinose Regulon in Bacillus subtilis by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping

The proteins involved in the utilization of L-arabinose by Bacillus subtilis are encoded by the araABDLMNPQ-abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene, and araR regulatory gene is induced by L-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within the araABDLMNPQ-abfA (OR(A1) and OR(A2)) and araE (OR(E1) and OR(E2)) promoters and noncooperatively to a single operator in the araR (OR(R3)) promoter region. Here, we have investigated how AraR controls transcription from the ara regulon in vivo. A deletion analysis of the ara promoters region showed that the five AraR binding sites are the key cis-acting regulatory elements of their corresponding genes. Furthermore, OR(E1)-OR(E2) and OR(R3) are auxiliary operators for the autoregulation of araR and the repression of araE, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the ara operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from the araABDLMNPQ-abfA operon and araE promoters, strongly suggesting that it is the major protein involved in the repression mechanism of L-arabinose-inducible expression in vivo. The ara regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.