Filipe Carvalho

In vivo transcriptional profiling of Listeria monocytogenes and mutagenesis identify new virulence factors involved in infection.

Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence.

The arsenal of virulence factors deployed by Listeria monocytogenes to promote its cell infection cycle

Listeria monocytogenes is an intracellular Gram-positive pathogen and the etiological agent of listeriosis, a human food-borne disease potentially fatal for certain risk groups. The virulence of L. monocytogenes is supported by a highly complex and coordinated intracellular life cycle that comprises several crucial steps: host cell adhesion and invasion, intracellular multiplication and motility, and intercellular spread. The completion of each stage is dependent on the orchestrated activity of specialized bacterial factors, in turn tightly controlled by a specific set of regulators. Some virulence factors and modulators also assume an important role in bacterial resistance and evasion to host defense mechanisms. In the last years, the advent of genomics promoted an increasingly prolific identification and functional characterization of new Listeria virulence factors. In this review, we summarize the current knowledge on nearly 50 molecules deployed by L. monocytogenes to promote its cell infection cycle.

Thymulin inhibits monocrotaline-induced pulmonary hypertension modulating interleukin-6 expression and suppressing p38 pathway

The pathogenesis of pulmonary hypertension (PH) includes an inflammatory response. Thymulin, a zinc-dependent thymic hormone, has important immunobiological effects by inhibiting various proinflammatory cytokines and chemokines. We investigated morphological and hemodynamic effects of thymulin administration in a rat model of monocrotaline (MCT)-induced PH, as well as the pattern of proinflammatory cytokine gene expression and the intracellular pathways involved. Adult Wistar rats received an injection of MCT (60 mg/kg, sc) or an equal volume of saline. One day after, the animals randomly received during 3 wk an injection of saline, vehicle (zinc plus carboxymethyl cellulose), or thymulin (100 ng/kg, sc, daily). At d 23-25, the animals were anesthetized for hemodynamic recordings, whereas heart and lungs were collected for morphometric and molecular analysis. Thymulin prevented morphological, hemodynamic, and inflammatory cardiopulmonary profile characteristic of MCT-induced PH, whereas part of these effects were also observed in MCT-treated animals injected with the thymulins vehicle containing zinc. The pulmonary thymulin effect was likely mediated through suppression of p38 pathway.

L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane.

Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for L-rhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection.

How Listeria monocytogenes organizes its surface for virulence.

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work “behind the frontline”, either supporting virulence effectors or ensuring the survival of the bacterium within its host.

PCR-based screening of targeted mutants for the fast and simultaneous identification of bacterial virulence factors.

Understanding the strategies used by pathogens to infect, survive, and proliferate in their hosts requires the identification of virulence factors. We developed PCR-based screening of targeted mutants to facilitate quick, simultaneous detection of multiple novel bacterial virulence genes. Based on direct PCR screening of pooled targeted mutants, this approach provides a fast and sensitive measure of virulence attenuation while significantly reducing the number of animals and time required. We demonstrate that the careful design of gene-specific primers allows the direct relative quantification of mixed mutants in infected mouse organs. Indeed, we show that the band intensity of the PCR product is directly related to the quantity of the corresponding strain in a pool of mutants. We applied the PCR-based screening of targeted mutants to the murine model of listeriosis and revealed new genes required for full pathogenicity of Listeria monocytogenes, a facultative human intracellular pathogen. PCR-based screening is a simple, useful, and fast technique to test pools of targeted bacterial mutants in vivo, without the requirements for a rigorous purification step, complicated PCR set-up, or special equipment. This approach can be adapted to other bacterial systems, constituting a significant advance in the field of infection biology.

Minimally Invasive Repair of Morgagni Hernia - A Multicenter Case Series

Children may benefit from minimally invasive surgery (MIS) in the correction of Morgagni hernia (MH). The present study aims to evaluate the outcome of MIS through a multicenter study. National institutions that use MIS in the treatment of MH were included. Demographic, clinical and operative data were analyzed. Thirteen patients with MH (6 males) were operated using similar MIS technique (percutaneous stitches) at a mean age of 22.2±18.3 months. Six patients had chromosomopathies (46%), five with Down syndrome (39%). Respiratory complaints were the most common presentation (54%). Surgery lasted 95±23min. In none of the patients was the hernia sac removed; prosthesis was never used. In the immediate post-operative period, 4 patients (36%) were admitted to intensive care unit (all with Down syndrome); all patients started enteral feeds within the first 24h. With a mean follow-up of 56±16.6 months, there were two recurrences (18%) at the same institution, one of which was repaired with an absorbable suture; both with Down syndrome. The application of MIS in the MH repair is effective even in the presence of comorbidities such as Down syndrome; the latter influences the immediate postoperative recovery and possibly the recurrence rate. Removal of hernia sac does not seem necessary. Non-absorbable sutures may be more appropriate.

Listeria monocytogenes encodes a functional ESX-1 secretion system whose expression is detrimental to in vivo infection.

ABSTRACT Bacterial pathogenicity deeply depends on the ability to secrete virulence factors that bind specific targets on host cells and manipulate host responses. The Gram-positive bacterium Listeria monocytogenes is a human foodborne pathogen that remains a serious public health concern. To transport proteins across its cell envelope, this facultative intracellular pathogen engages a set of specialized secretion systems. Here we show that L. monocytogenes EGDe uses a specialized secretion system, named ESX-1, to secrete EsxA, a homolog of the virulence determinants ESAT-6 and EsxA of Mycobacterium tuberculosis and Staphylococcus aureus, respectively. Our data show that the L. monocytogenes ESX-1 secretion system and its substrates are dispensable for bacterial invasion and intracellular multiplication in eukaryotic cell lines. Surprisingly, we found that the EssC-dependent secretion of EsxA has a detrimental effect on L. monocytogenes in vivo infection.

Occurrence of mutations impairing sigma factor B (SigB) function upon inactivation of Listeria monocytogenes genes encoding surface proteins.

Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent to which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we used proteomics to analyse mutants of the intracellular pathogen Listeria monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions, such as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried loss-of-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in a large series of new inlG or vip mutants constructed for this purpose were, however, unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using common genetic procedures or during subculturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components.

The Tat Pathway Is Prevalent in Listeria monocytogenes Lineage II and Is Not Required for Infection and Spread in Host Cells

Listeria monocytogenes, a foodborne pathogenic bacterium, remains a serious public health concern due to its frequent occurrence in food products coupled with a high mortality rate. Bacterial pathogenicity depends greatly on the ability to secrete virulence factors to or beyond the bacterial cell surface. The Tat pathway, one of the secretion systems present in L. monocytogenes, was until now only investigated in silico. In L. monocytogenes strain EGDe two genes constitute this pathway, tatC(lmo0361) and tatA(lmo0362). Here we show that tatC and tatA are cotranscribed in a bicistronic- and growth-phase-dependent manner, being downregulated in the stationary phase. An EGDe tatAC mutant strain (EGDe ΔtatAC) was constructed, confirming that the Tat pathway is not essential for L.monocytogenes survival or biofilm-forming ability. When compared to the wild-type EGDe, deletion of tatAC did not decrease the virulence potential of EGDe ΔtatAC in HT-29 human epithelial cell line and even increased (p < 0.05) the virulence potential for mice. Moreover, we show that tat genes are prevalent in L. monocytogenes strains belonging to genetic lineage II and are generally absent from lineage I, which is more associated with human cases, thus excluding the possibility of using the Tat system as a target for novel antimicrobial compounds targeting L.monocytogenes.