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Infection Biology lab

Our general goal

The main interest of the laboratory is to understand molecular and cellular mechanisms underlying bacterial virulence. In particular, we study processes by which bacterial pathogens alter the normal functioning of eukaryotic host cells.

Our favourite virulence mechanism

We focus in one mechanism in particular, named type III secretion (T3S), which is used by Gram-negative bacteria to deliver - in one single step - effector proteins into eukaryotic cells (Figure 1). T3S systems are found in a wide variety of pathogenic bacteria, for animals and plants, and also in symbionts. The effectors act on a vast array of cell functions, such as cytoskeleton dynamics, cell signalling, membrane trafficking, apoptosis, or cell cycle. The T3S effectors are eukaryotic-like proteins because, in activity, function, or structure, they often resemble proteins found in higher organisms.

 

Figure 1. Type III Secretion. Simplified representation of the protein transport mechanism of type III secretion, by which bacteria using a sophisticated apparatus - known as an injectisome, which in many cases is seen in electron microcraphs as a needle-like structure protruding from the bacterial surface - injects effectors into eukaryotic host cells through a translocon pore that is thought to physically link the injectisome needle to a host cell lipid membrane. This enables the delivery of the effector proteins into host cells in a single step. The length of the injectisome needle is tighly controlled by a protein functioning as a molecular ruler. This tight control is needed to overcome the length of other structures at the bacterial surface (e.g. adhesins or LPS) and to enable host cell sensing by the needle tip. Recent years witnessed huge progresses in our knowledge of the structure of the bacterial injectisome.

Our bugs

We focus on intracellular bacterial pathogens that multiply within host cells in unique membrane-bound organelles (pathogen-containing vacuoles), using Chlamydia trachomatis and Salmonella enterica as experimental models (Figure 2).

Intravacuolar pathogens.jpg

Figure 2. Intravacuolar bacterial pathogens. Chlamydia and Salmonella use T3S systems to invade and replicate within host cells. Please note that while C. trachomatis resides within a large and unique vacuole (as shown), known as an inclusion, Salmonella is normally seen within host cells in single individual vacuoles (not shown).

 

Chlamydia trachomatis

C. trachomatis is part of a large group of highly related Gram-negative bacteria that are characterized by their obligate growth within eukaryotic cells and which includes the etiological agents of many important human and animal diseases. C. trachomatis causes ocular and genital infections in humans. The ocular serovars infect the conjuctival epithelium and can give rise to trachoma, the leading cause of preventable blindness in developing countries. The genital serovars are the most frequent bacterial cause of sexually transmitted disease, worldwide. The genital infection is often asymptomatic and if left untreated can cause inflammatory diseases which, in turn, can lead to infertility and ectopic pregnancies in women.

Chlamydiae are characterised by a unique developmental/infectious cycle that includes two morphological forms: the infectious but metabolically inert elementary bodies (EB), and the non-infectious but metabolically active reticulate bodies (RBs) (Figure 3) Relatively little is known about the mechanisms by which chlamydiae manipulates host cells because they have been intractable to genetic manipulation. Only very recently breakthroughs were reported with this respect in PNAS and PLoS Pathogens. However, all chlamydiae code for the core components of a T3S system. It is predicted that chlamydial T3S effector proteins play crucial roles throughout the chlamydial developmental/infectious cycle. This includes proteins translocated across the plasma membrane - e.g. to allow entry of EBs - as well as proteins translocated across the membrane of the large vacuole (known as inclusion) that encloses EBs and RBs.

Figure 3. Chlamydiae developmental/infectious cycle (image from Sara Pais). Chlamydial elementary bodies (EBs; green) are believed to be packed with T3S effectors and have an assembled injectisome. Upon cell attachment, the effectors (e.g. TARP and CT694) are delivered into host cells, which is thought to mediate the invasion process. Upon internalization, EBs reside in a membrane bound vacuole and rapidly differentiate into reticulate bodies (RBs; yellow). The initially plasma membrane-derived vacuole is modified by Chlamydia through the T3S of Inclusion membrane proteins (Incs), which decorate the inclusion membrane throughout the cycle. The RBs replicate by binary fission up to a point where bacterial replication becomes asynchronous and yields both RBs and EBs. Finally, when the inclusion becomes filled with EBs, lysis or extrusion of the host cell occurs and the EBs can start another round of infection.

Chlamydial T3S effectors are thought to modulate the host cell throughout the developmental/infectious cycle (e.g. modification of the inclusion to avoid phagolysosomal fusion, interference with membrane trafficking for acquisition of nutrients and membrane, or inhibition of apoptosis). It is noteworthy that Chlamydia uses other mechanisms other than T3S to deliver effector proteins into host cells. For example, CPAF, a critical chlamydial effector protein, is not a T3S substrate; yet, how CPAF is translocated across the inclusion membrane is unknown

 

Salmonella enterica

Salmonella infections remain important causes of human morbidity and mortality and are a significant public health concern. S. enterica serovar Typhimurium (S. Typhimurium; a leading cause of human gastroenteritis) has been extensively used to study mechanisms underlying Salmonella virulence, because of the availability of sophisticated genetic tools; of excellent tissue culture models; and of a mice model of typhoid fever (caused by S. Typhi). S. Typhimurium is also an excellent model to analyse basic mechanisms of host-pathogen interactions, such as invasion and intracellular replication within host cells.

S. enterica are facultative intracellular bacteria which proliferate within host cells in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). Host cell invasion by Salmonella is largely dependent on the extracellularly expressed Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS-1). Within host cells, intracellular Salmonella activate T3SS-2, which translocates over 20 T3S effectors across the SCV membrane. The collective action of T3SS-2 effectors promotes intracellular bacterial replication (Figure 4). 

 

Figure 4. Scheme of an infection of epithelial cells by Salmonella enterica and temporal and spatial expression of T3SS-1 and T3SS-2.

T3SS-2 effectors might promote bacterial intracellular replication by direct manipulation of host cell membrane trafficking and molecular motors, such as cytoplasmic dynein, kinesin-1, and myosin II. This particular T3SS-2-dependent manipulation of host cells has a read-out in HeLa cells infected with S. Typhimurium in form of the apperance of tight clusters (microcolonies) of SCVs in a Golgi/perinuclear region and of dramatic Salmonella-induced membrane tubules enriched in host cell proteins (e.g. LAMP1 and SCAMP3) and in T3SS-2 effectors (Figure 5).

 

Figure 5. Salmonella-induced membrane tubules. HeLa cells infected with S. Typhimurium sseF- + pSseF-HA for 14 h and immunolabelled for LAMP1 (blue), SseF-HA (green), and SCAMP3 (red).

Our research projects

The major specific aim of our lab is to understand the function of individual T3S effectors of Chlamydia and Salmonella. We believe that this will contribute to further our knowledge of the virulence mechanisms used by these bacteria, and could provide novel insights into the cell biology of eukaryotic cells or reveal novel drug targets. In addition, we are also studying T3S chaperones of C. trachomatis.

We are well aware that effectors delivered by a single bacterium into a host cell act together and often have redundant functions, and thus analyses of the function of single effectors can be misleading and extremely difficult. However, we believe that continuous efforts - from different groups - to understand the molecular and cellular function of single effectors will eventually provide a comprehensive picture of virulence mechanisms of bacteria injecting host cells with T3S effectors.
 

Project 1. Identification and characterisation of novel C. trachomatis T3S effectors
It has been estimated that C. trachomatis may deliver > 100 effectors into host cells. While in recent years we witnessed the discovery of a considerable number of novel chlamydial T3S effectors (TARP/CT456, NUE/CT737, CT694, CT695, CT847, CT619, CT620, CT621, CT711, CT712), it is likely that many others remain unidentified. To search for candidate novel effectors of C. trachomatis, we have searched its genome for genes encoding hypothetical proteins with no predictable function and which have not been studied before. Then, to test if the selected genes are T3S substrates, we used Yersinia enterocolitica as heterologous bacteria assembling a T3S system that is able to recognise chlamydial T3S substrates, a methodology originally described by Agathe Subtil and Ken Fields. We are currently i) testing if the candidate C. trachomatis T3S substrates found are secreted in Chlamydia-infected cells, and ii) performing functional screens with the candidate effectors in mammalian cells and in the yeast Saccharomyces cerevisiae (an eukaryotic model organism).


Project 2. Functional analyses of Inc proteins of C. trachomatis
Incs are a large group of chlamydiae-specific proteins (representing about 5 % of the coding capacity of chlamydial genomes) and play an important role in Chlamydiae-host cell interactions. For example, the genome of C. trachomatis encodes for > 50 Incs.  Inc proteins share a unique hydrophobic motif thought to target them to the inclusion membrane and are believed to be T3S substrates. However, as yet, this has not been shown for all Incs and, except for a few notable cases, their biological roles remain largely unknown (but Incs with known function seem to modulate host cell transport pathways). We are i) using bioinformatics and molecular biology methods to study phylogeny, evolution and regulation of expression of inc genes relative to the specificities of C. trachomatis infections (F. Almeida, V. Borges et al, manuscript in preparation), and ii) molecular cell biology approaches to understand the function of specific and rationally selected Incs.


Project 3. Identification and characterisation of novel C. trachomatis T3S effector-chaperone pairs

Secretion of T3S substrates often involves characteristic T3S chaperones - with a low molecular weight, an acidic pI, and which form dimers, and do not bind or hydrolyze ATP - that often cover membrane localisation domains in the effectors. Surprisingly, although in recent years the number of known chlamydial T3S effectors has been increasing, little is known about their possible cognate T3S chaperones. To date, only two effector chaperones (CT043/Slc1 and CT260/Mcsc) have been identified. We are searching for novel C. trachomatis T3S effector-chaperone pairs by using bioinformatics and molecular microbiology approaches. We hope to gain insights on how Chlamydia regulates the secretion of possibly > 100 T3S substrates during its developmental/infectious cycle.


Project 4. Analyses of the function of poorly characterised Salmonella T3SS-2 effectors
In general, the biological role and biochemical activity of T3SS-2 effectors are not fully characterised. We focus on poorly characterised T3SS-2 effectors and use molecular and cellular microbiology methods aiming to understand the mode of action of the effectors. We are particularly interested in the mechanisms by which T3SS-2 effectors promote bacterial intracellular replication by modulation of host cell membrane trafficking and molecular motors, which is revealed in infected HeLa cells by the appearance of dramatic Salmonella-induced tubules and by specific SCV positioning and movement relative to host cell structures (MTOC/Golgi) and to other bacterial vacuoles.

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