The prostaglandin D2 antagonist asapiprant ameliorates clinical severity in young hosts infected with invasive Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) remains a serious cause of pulmonary and systemic infections globally, and host-directed therapies are lacking. The aim of this study was to test the therapeutic efficacy of asapiprant, an inhibitor of prostaglandin D2 signaling, against pneumococcal infection. Treatment of young mice with asapiprant after pulmonary infection with invasive pneumococci significantly reduced systemic spread, disease severity, and host death. Protection was specific against bacterial dissemination from the lung to the blood but had no effect on pulmonary bacterial burden. Asapiprant-treated mice had enhanced antimicrobial activity in circulating neutrophils, elevated levels of reactive oxygen species (ROS) in lung macrophages/monocytes, and improved pulmonary barrier integrity indicated by significantly reduced diffusion of fluorescein isothiocyanate (FITC)-dextran from lungs into the circulation. These findings suggest that asapiprant protects the host against pneumococcal dissemination by enhancing the antimicrobial activity of immune cells and maintaining epithelial/endothelial barrier integrity in the lungs.

Activating A1 adenosine receptor signaling boosts early pulmonary neutrophil recruitment in aged mice in response to Streptococcus pneumoniae infection.

Background: Streptococcus pneumoniae (pneumococcus) is a leading cause of pneumonia in older adults. Successful control of pneumococci requires robust pulmonary neutrophil influx early in infection. However, aging is associated with aberrant neutrophil recruitment and the mechanisms behind that are not understood. Here we explored how neutrophil recruitment following pneumococcal infection changes with age and the host pathways regulating this. Results: Following pneumococcal infection there was a significant delay in early neutrophil recruitment to the lungs of aged mice. Neutrophils from aged mice showed defects in trans-endothelial migration in vitro compared to young controls. To understand the pathways involved, we examined immune modulatory extracellular adenosine (EAD) signaling, that is activated upon cellular damage. Signaling through the lower affinity A2A and A2B adenosine receptors had no effect on neutrophil recruitment to infected lungs. In contrast, inhibition of the high affinity A1 receptor in young mice blunted neutrophil recruitment to the lungs following infection. A1 receptor inhibition decreased expression of CXCR2 on circulating neutrophils, which is required for transendothelial migration. Indeed, A1 receptor signaling on neutrophils was required for their ability to migrate across endothelial cells in response to infection. Aging was not associated with defects in EAD production or receptor expression on neutrophils. However, agonism of A1 receptor in aged mice rescued the early defect in neutrophil migration to the lungs and improved control of bacterial burden. Conclusions: This study suggests age-driven defects in EAD damage signaling can be targeted to rescue the delay in pulmonary neutrophil migration in response to bacterial pneumonia.

Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling.

Polymorphonuclear cells (PMNs) control Streptococcus pneumoniae (pneumococcus) infection through various antimicrobial activities. We previously found that reactive oxygen species (ROS) were required for optimal antibacterial function, however, the NADPH oxidase is known to be dispensable for the ability of PMNs to kill pneumococci. In this study, we explored the role of ROS produced by the mitochondria in PMN antimicrobial defense against pneumococci. We found that the mitochondria are an important source of overall intracellular ROS produced by murine PMNs in response to infection. We investigated the host and bacterial factors involved and found that mitochondrial ROS (MitROS) are produced independent of bacterial capsule or pneumolysin but presence of live bacteria that are in direct contact with PMNs enhanced the response. We further found that MyD88-/- PMNs produced less MitROS in response to pneumococcal infection suggesting that released bacterial products acting as TLR ligands are sufficient for inducing MitROS production in PMNs. To test the role of MitROS in PMN function, we used an opsonophagocytic killing assay and found that MitROS were required for the ability of PMNs to kill pneumococci. We then investigated the role of MitROS in host resistance and found that MitROS are produced by PMNs in response to pneumococcal infection. Importantly, treatment of mice with a MitROS scavenger prior to systemic challenge resulted in reduced survival of infected hosts. In exploring host pathways that control MitROS, we focused on extracellular adenosine, which is known to control PMN anti-pneumococcal activity, and found that signaling through the A2B adenosine receptor inhibits MitROS production by PMNs. A2BR-/- mice produced more MitROS and were significantly more resistant to infection. Finally, we verified the clinical relevance of our findings using human PMNs. In summary, we identified a novel pathway that controls MitROS production by PMNs, shaping host resistance against S. pneumoniae.

The Age-Driven Decline in Neutrophil Function Contributes to the Reduced Efficacy of the Pneumococcal Conjugate Vaccine in Old Hosts.

Despite the availability of vaccines, Streptococcus pneumoniae (pneumococcus) remains a serious cause of infections in the elderly. The efficacy of anti-pneumococcal vaccines declines with age. While age-driven changes in antibody responses are well defined, less is known about the role of innate immune cells such as polymorphonuclear leukocytes (PMNs) in the reduced vaccine protection seen in aging. Here we explored the role of PMNs in protection against S. pneumoniae in vaccinated hosts. We found that depletion of PMNs in pneumococcal conjugate vaccine (PCV) treated young mice prior to pulmonary challenge with S. pneumoniae resulted in dramatic loss of host protection against infection. Immunization boosted the ability of PMNs to kill S. pneumoniae and this was dependent on bacterial opsonization by antibodies. Bacterial opsonization with immune sera increased several PMN anti-microbial activities including bacterial uptake, degranulation and ROS production. As expected, PCV failed to protect old mice against S. pneumoniae. In probing the role of PMNs in this impaired protection, we found that aging was accompanied by an intrinsic decline in PMN function. PMNs from old mice failed to effectively kill S. pneumoniae even when the bacteria were opsonized with immune sera from young controls. In exploring mechanisms, we found that PMNs from old mice produced less of the antimicrobial peptide CRAMP and failed to efficiently kill engulfed pneumococci. Importantly, adoptive transfer of PMNs from young mice reversed the susceptibility of vaccinated old mice to pneumococcal infection. Overall, this study demonstrates that the age-driven decline in PMN function impairs vaccine-mediated protection against Streptococcus pneumoniae.

Intranasal Vaccine Delivery Technology for Respiratory Tract Disease Application with a Special Emphasis on Pneumococcal Disease.

This mini-review will cover recent trends in intranasal (IN) vaccine delivery as it relates to applications for respiratory tract diseases. The logic and rationale for IN vaccine delivery will be compared to methods and applications accompanying this particular administration route. In addition, we will focus extended discussion on the potential role of IN vaccination in the context of respiratory tract diseases, with a special emphasis on pneumococcal disease. Here, elements of this disease, including its prevalence and impact upon the elderly population, will be viewed from the standpoint of improving health outcomes through vaccine design and delivery technology and how IN administration can play a role in such efforts.

Transcriptome Profiling Reveals CD73 and Age-Driven Changes in Neutrophil Responses against Streptococcus pneumoniae.

Neutrophils are required for host resistance against Streptococcus pneumoniae, but their function declines with age. We previously found that CD73, an enzyme required for antimicrobial activity, is downregulated in neutrophils (also known as polymorphonuclear leukocytes [PMNs]) from aged mice. This study explored transcriptional changes in neutrophils induced by S. pneumoniae to identify pathways controlled by CD73 and dysregulated with age. Pure bone marrow-derived neutrophils isolated from wild-type (WT) young and old and CD73 knockout (CD73KO) young mice were mock challenged or infected with S. pneumoniae ex vivo. RNA sequencing (RNA-Seq) was performed to identify differentially expressed genes (DEGs). We found that infection triggered distinct global transcriptional changes across hosts that were strongest in CD73KO neutrophils. Surprisingly, there were more downregulated than upregulated genes in all groups upon infection. Downregulated DEGs indicated a dampening of immune responses in old and CD73KO hosts. Further analysis revealed that CD73KO neutrophils expressed higher numbers of long noncoding RNAs (lncRNAs) than those in WT controls. Predicted network analysis indicated that CD73KO-specific lncRNAs control several signaling pathways. We found that genes in the c-Jun N-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) pathway were upregulated upon infection in CD73KO mice and in WT old mice, but not in WT young mice. This corresponded to functional differences, as phosphorylation of the downstream AP-1 transcription factor component c-Jun was significantly higher in neutrophils from infected CD73KO mice and old mice. Importantly, inhibition of JNK/AP-1 rescued the ability of these neutrophils to kill S. pneumoniae. Together, our findings revealed that the ability of neutrophils to modify their gene expression to better adapt to bacterial infection is in part regulated by CD73 and declines with age.

Older but Not Wiser: the Age-Driven Changes in Neutrophil Responses during Pulmonary Infections.

Elderly individuals are at increased risk of life-threatening pulmonary infections. Neutrophils are a key determinant of the disease course of pathogen-induced pneumonia. Optimal host defense balances initial robust pulmonary neutrophil responses to control pathogen numbers, ultimately followed by the resolution of inflammation to prevent pulmonary damage. Recent evidence suggests that phenotypic and functional heterogeneity in neutrophils impacts host resistance to pulmonary pathogens. Apart from their apparent role in innate immunity, neutrophils also orchestrate subsequent adaptive immune responses during infection. Thus, the outcome of pulmonary infections can be shaped by neutrophils. This review summarizes the age-driven impairment of neutrophil responses and the contribution of these cells to the susceptibility of the elderly to pneumonia. We describe how aging is accompanied by changes in neutrophil recruitment, resolution, and function. We discuss how systemic and local changes alter the neutrophil phenotype in aged hosts. We highlight the gap in knowledge of whether these changes in neutrophils also contribute to the decline in adaptive immunity seen with age. We further detail the factors that drive dysregulated neutrophil responses in the elderly and the pathways that may be targeted to rebalance neutrophil activity and boost host resistance to pulmonary infections.

Liposomal Encapsulation of Polysaccharides (LEPS) as an Effective Vaccine Strategy to Protect Aged Hosts Against S. pneumoniae Infection.

Despite the availability of licensed vaccines, pneumococcal disease caused by the bacteria Streptococcus pneumoniae (pneumococcus), remains a serious infectious disease threat globally. Disease manifestations include pneumonia, bacteremia, and meningitis, resulting in over a million deaths annually. Pneumococcal disease disproportionally impacts older adults aged ≥65 years. Interventions are complicated through a combination of complex disease progression and 100 different bacterial capsular polysaccharide serotypes. This has made it challenging to develop a broad vaccine against S. pneumoniae, with current options utilizing capsular polysaccharides as the primary antigenic content. However, current vaccines are substantially less effective in protecting the elderly. We previously developed a Liposomal Encapsulation of Polysaccharides (LEPS) vaccine platform, designed around limitations of current pneumococcal vaccines, that allowed the noncovalent coupling of polysaccharide and protein antigen content and protected young hosts against pneumococcal infection in murine models. In this study, we modified the formulation to make it more economical and tested the novel LEPS vaccine in aged hosts. We found that in young mice (2-3 months), LEPS elicited comparable responses to the pneumococcal conjugate vaccine Prevnar-13. Further, LEPS immunization of old mice (18-22 months) induced comparable antibody levels and improved antibody function compared to Prevnar-13. Importantly, LEPS protected old mice against both invasive and lung localized pneumococcal infections. In summary, LEPS is an alternative and effective vaccine strategy that protects aged hosts against different manifestations of pneumococcal disease.

Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary Neutrophils.

Antibodies against Streptococcus pneumoniae (pneumococcus) following vaccination are crucial for host protection against invasive pneumococcal infections. The antibodies induced by pneumococcal vaccines act as opsonins to mediate bacterial uptake and killing by host phagocytic cells, especially polymorphonuclear leukocytes (PMNs) also called neutrophils. Therefore, it is important to measure not only the levels of antibodies induced by a pneumococcal vaccine candidate but their actual functional capacity in mediating bacterial opsonization and killing by PMNs. Here, we describe a protocol to demonstrate effective deposition of vaccine-induced antibodies on the surface of S. pneumoniae by flow cytometry and subsequent opsonophagocytic killing (OPH) by murine bone-marrow derived PMNs.

Extracellular adenosine signaling reverses the age-driven decline in the ability of neutrophils to kill Streptococcus pneumoniae.

The elderly are susceptible to serious infections by Streptococcus pneumoniae (pneumococcus), which calls for a better understanding of the pathways driving the decline in host defense in aging. We previously found that extracellular adenosine (EAD) shaped polymorphonuclear cell (PMN) responses, which are crucial for controlling infection. EAD is produced by CD39 and CD73, and signals via A1, A2A, A2B, and A3 receptors. The objective of this study was to explore the age-driven changes in the EAD pathway and its impact on PMN function. We found in comparison to young mice, PMNs from old mice expressed significantly less CD73, but similar levels of CD39 and adenosine receptors. PMNs from old mice failed to efficiently kill pneumococci ex vivo; however, supplementation with adenosine rescued this defect. Importantly, transfer of PMNs expressing CD73 from young mice reversed the susceptibility of old mice to pneumococcal infection. To identify which adenosine receptor(s) is involved, we used specific agonists and inhibitors. We found that A1 receptor signaling was crucial for PMN function as inhibition or genetic ablation of A1 impaired the ability of PMNs from young mice to kill pneumococci. Importantly, activation of A1 receptors rescued the age-associated defect in PMN function. In exploring mechanisms, we found that PMNs from old mice failed to efficiently kill engulfed pneumococci and that A1 receptor controlled intracellular killing. In summary, targeting the EAD pathway reverses the age-driven decline in PMN antimicrobial function, which has serious implications in combating infections.

Neutrophils Are Required During Immunization With the Pneumococcal Conjugate Vaccine for Protective Antibody Responses and Host Defense Against Infection.

Neutrophils can shape adaptive immunity; however, their role in vaccine-induced protection against infections in vivo remains unclear. Here, we tested their role in the clinically relevant polysaccharide conjugate vaccine against Streptococcus pneumoniae (pneumococcus). We antibody depleted neutrophils during vaccination, allowed them to recover, and 4 weeks later challenged mice with pneumococci. We found that while isotype-treated vaccinated controls were protected against an otherwise lethal infection in naive mice, full protection was lost upon neutrophil depletion. Compared to vaccinated controls, neutrophil-depleted mice had higher lung bacterial burdens, increased incidence of bacteremia, and lower survival rates. Sera from neutrophil-depleted mice had less antipneumococcal IgG2c and IgG3, were less efficient at inducing opsonophagocytic killing of bacteria by neutrophils in vitro, and were worse at protecting naive mice against pneumococcal pneumonia. In summary, neutrophils are required during vaccination for optimal host protection, which has important implications for future vaccine design against pneumococci and other pathogens.

Listeria monocytogenes exploits host exocytosis to promote cell-to-cell spread.

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.

Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production.

Polymorphonuclear leukocytes (PMNs) are crucial for initial control of Streptococcus pneumoniae (pneumococcus) lung infection; however, as the infection progresses their persistence in the lungs becomes detrimental. Here we explored why the antimicrobial efficacy of PMNs declines over the course of infection. We found that the progressive inability of PMNs to control infection correlated with phenotypic differences characterized by a decrease in CD73 expression, an enzyme required for production of extracellular adenosine (EAD). EAD production by CD73 was crucial for the ability of both murine and human PMNs to kill S. pneumoniae. In exploring the mechanisms by which CD73 controlled PMN function, we found that CD73 mediated its antimicrobial activity by inhibiting IL-10 production. PMNs from wild-type mice did not increase IL-10 production in response to S. pneumoniae; however, CD73-/- PMNs up-regulated IL-10 production upon pneumococcal infection in vitro and during lung challenge. IL-10 inhibited the ability of WT PMNs to kill pneumococci. Conversely, blocking IL-10 boosted the bactericidal activity of CD73-/- PMNs as well as host resistance of CD73-/- mice to pneumococcal pneumonia. CD73/IL-10 did not affect apoptosis, bacterial uptake, and intracellular killing or production of antimicrobial neutrophil elastase and myeloperoxidase. Rather, inhibition of IL-10 production by CD73 was important for optimal reactive oxygen species (ROS) production by PMNs. ROS contributed to PMN antimicrobial function as their removal or detoxification impaired the ability of PMNs to efficiently kill S. pneumoniae. This study demonstrates that CD73 controls PMN antimicrobial phenotype during S. pneumoniae infection.

A1 adenosine receptor signaling reduces Streptococcus pneumoniae adherence to pulmonary epithelial cells by targeting expression of platelet-activating factor receptor.

Extracellular adenosine production is crucial for host resistance against Streptococcus pneumoniae (pneumococcus) and is thought to affect antibacterial immune responses by neutrophils. However, whether extracellular adenosine alters direct host-pathogen interaction remains unexplored. An important determinant for lung infection by S. pneumoniae is its ability to adhere to the pulmonary epithelium. Here we explored whether extracellular adenosine can directly impact bacterial adherence to lung epithelial cells. We found that signaling via A1 adenosine receptor significantly reduced the ability of pneumococci to bind human pulmonary epithelial cells. A1 receptor signaling blocked bacterial binding by reducing the expression of platelet-activating factor receptor, a host protein used by S. pneumoniae to adhere to host cells. In vivo, A1 was required for control of pneumococcal pneumonia as inhibiting it resulted in increased host susceptibility. As S. pneumoniae remain a leading cause of community-acquired pneumonia in the elderly, we explored the role of A1 in the age-driven susceptibility to infection. We found no difference in A1 pulmonary expression in young versus old mice. Strikingly, triggering A1 signaling boosted host resistance of old mice to S. pneumoniae pulmonary infection. This study demonstrates a novel mechanism by which extracellular adenosine modulates resistance to lung infection by targeting bacterial-host interactions.

The Host Scaffolding Protein Filamin A and the Exocyst Complex Control Exocytosis during InlB-Mediated Entry of Listeria monocytogenes.

Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry, in part, through stimulation of localized exocytosis. How exocytosis is upregulated during entry is not understood. Here, we show that the human signaling proteins mTOR, protein kinase C-α (PKC-α), and RalA promote exocytosis during entry by controlling the scaffolding protein Filamin A (FlnA). InlB-mediated uptake was accompanied by PKC-α-dependent phosphorylation of serine 2152 in FlnA. Depletion of FlnA by RNA interference (RNAi) or expression of a mutated FlnA protein defective in phosphorylation impaired InlB-dependent internalization. These findings indicate that phosphorylation of FlnA by PKC-α contributes to entry. mTOR and RalA were found to mediate the recruitment of FlnA to sites of InlB-mediated entry. Depletion of PKC-α, mTOR, or FlnA each reduced exocytosis during InlB-mediated uptake. Because the exocyst complex is known to mediate polarized exocytosis, we examined if PKC-α, mTOR, RalA, or FlnA affects this complex. Depletion of PKC-α, mTOR, RalA, or FlnA impaired recruitment of the exocyst component Exo70 to sites of InlB-mediated entry. Experiments involving knockdown of Exo70 or other exocyst proteins demonstrated an important role for the exocyst complex in uptake of Listeria Collectively, our results indicate that PKC-α, mTOR, RalA, and FlnA comprise a signaling pathway that mobilizes the exocyst complex to promote infection by Listeria.

Interaction of microbial pathogens with host exocytic pathways.

Many microbial pathogens co-opt or perturb host membrane trafficking pathways. This review covers recent examples in which microbes interact with host exocytosis, the fusion of intracellular vesicles with the plasma membrane. The bacterial pathogens Listeria monocytogenes and Staphylococcus aureus subvert recycling endosomal pathways of exocytosis in order to induce their entry into human cells. By contrast, entry of the protozoan pathogen Trypanosoma cruzi or the virus adenovirus into host cells involves exploitation of lysosomal exocytosis. Toxins produced by Bacillus anthracis or Vibrio cholerae interfere with exocytosis pathways mediated by the GTPase Rab11 and the exocyst complex. By doing so, anthrax or cholera toxins impair recycling of cadherins to cell-cell junctions and disrupt the barrier properties of endothelial cells or intestinal epithelial cells, respectively. Uropathogenic Escherichia coli (UPEC) is expelled from bladder epithelial cells through two different exocytic routes that involve sensing of bacteria in vacuoles by host Toll-like receptor 4 (TLR4) or monitoring of the pH of lysosomes harbouring UPEC. The TLR4 pathway is mediated by multiple Rab GTPases and the exocyst, whereas the other pathway involves exocytosis of lysosomes. Expulsion of UPEC through these pathways is thought to benefit the host.

A role for host cell exocytosis in InlB-mediated internalisation of Listeria monocytogenes.

The bacterial surface protein InlB mediates internalisation of Listeria monocytogenes into human cells through interaction with the host receptor tyrosine kinase, Met. InlB-mediated entry requires localised polymerisation of the host actin cytoskeleton. Apart from actin polymerisation, roles for other host processes in Listeria entry are unknown. Here, we demonstrate that exocytosis in the human cell promotes InlB-dependent internalisation. Using a probe consisting of VAMP3 with an exofacial green fluorescent protein tag, focal exocytosis was detected during InlB-mediated entry. Exocytosis was dependent on Met tyrosine kinase activity and the GTPase RalA. Depletion of SNARE proteins by small interfering RNA demonstrated an important role for exocytosis in Listeria internalisation. Depletion of SNARE proteins failed to affect actin filaments during internalisation, suggesting that actin polymerisation and exocytosis are separable host responses. SNARE proteins were required for delivery of the human GTPase Dynamin 2, which promotes InlB-mediated entry. Our results identify exocytosis as a novel host process exploited by Listeria for infection.

Host Serine/Threonine Kinases mTOR and Protein Kinase C-α Promote InlB-Mediated Entry of Listeria monocytogenes.

The bacterial pathogen Listeria monocytogenes causes foodborne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some human cells through interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met. InlB-dependent entry requires localized polymerization of the host actin cytoskeleton. The signal transduction pathways that act downstream of Met to regulate actin filament assembly or other processes during Listeria uptake remain incompletely characterized. Here, we demonstrate important roles for the human serine/threonine kinases mTOR and protein kinase C-α (PKC-α) in InlB-dependent entry. Experiments involving RNA interference (RNAi) indicated that two multiprotein complexes containing mTOR, mTORC1 and mTORC2, are each needed for efficient internalization of Listeria into cells of the human cell line HeLa. InlB stimulated Met-dependent phosphorylation of mTORC1 or mTORC2 substrates, demonstrating activation of both mTOR-containing complexes. RNAi studies indicated that the mTORC1 effectors 4E-BP1 and hypoxia-inducible factor 1α (HIF-1α) and the mTORC2 substrate PKC-α each control Listeria uptake. Genetic or pharmacological inhibition of PKC-α reduced the internalization of Listeria and the accumulation of actin filaments that normally accompanies InlB-mediated entry. Collectively, our results identify mTOR and PKC-α to be host factors exploited by Listeria to promote infection. PKC-α controls Listeria entry, at least in part, by regulating the actin cytoskeleton downstream of the Met receptor.

Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica.

Many bacterial pathogens subvert mammalian type IA phosphoinositide 3-kinase (PI3K) in order to induce their internalization into host cells. How PI3K promotes internalization is not well understood. Also unclear is whether type IA PI3K affects different pathogens through similar or distinct mechanisms. Here, we performed an RNA interference (RNAi)-based screen to identify components of the type IA PI3K pathway involved in invasin-mediated entry of Yersinia enterocolitica, an enteropathogen that causes enteritis and lymphadenitis. The 69 genes targeted encode known upstream regulators or downstream effectors of PI3K. A similar RNAi screen was previously performed with the food-borne bacterium Listeria monocytogenes The results of the screen with Y. enterocolitica indicate that at least nine members of the PI3K pathway are needed for invasin-mediated entry. Several of these proteins, including centaurin-α1, Dock180, focal adhesion kinase (FAK), Grp1, LL5α, LL5ß, and PLD2 (phospholipase D2), were recruited to sites of entry. In addition, centaurin-α1, FAK, PLD2, and mTOR were required for remodeling of the actin cytoskeleton during entry. Six of the human proteins affecting invasin-dependent internalization also promote InlB-mediated entry of L. monocytogenes Our results identify several host proteins that mediate invasin-induced effects on the actin cytoskeleton and indicate that a subset of PI3K pathway components promote internalization of both Y. enterocolitica and L. monocytogenes.

Inhibition of endotoxin-induced hepatotoxicity by melatonin in rats.

Bacterial endotoxin or lipopolysaccharide causes extensive damage to various organs including the liver. This is due to an increased production of tumor necrosis factor α induced- reactive intermediates. These intermediates are known to cause extensive damage to a variety of cellular biomolecules leading to oxidative stress. In the present study, the role of the pineal hormone melatonin was evaluated as an antioxidant against endotoxin induced- hepatotoxicity using Wistar rats. Bacterial endotoxin was injected (i.v) and animals were sacrificed 8h post-challenge. Endotoxemia was associated with a statistically significant rise in the serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and also caused histopathological changes. Administration of melatonin could significantly attenuate these enzymatic and associated histological alterations. Melatonin was administered (i.p) pre and/or post endotoxin challenge. A significant reduction in the levels of malondialdehyde and tumor necrosis factor-α in the hepatic tissue was also observed with melatonin supplementation. Reduction in the levels of endogenous antioxidants such as superoxide dismutase, catalase and reduced glutathione after endotoxin challenge was effectively attenuated by the administration of melatonin. Endotoxin challenge caused a marked increases in the levels of nitrite, and this was significantly lowered by melatonin administration. The above mentioned changes might have resulted in endotoxin associated hepatocellular necrosis which was minimized by melatonin supplementation in the present study.