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Capsule prolongs survival of Streptococcus pneumoniae during starvation

Hamaguchi, Shigeto; Zafar, M Ammar; Cammer, Michael; Weiser, Jeffrey N
Person-to-person transmission of Streptococcus pneumoniae (the pneumococcus) may occur via environmental sources in close contact with carriers. Pneumococcal polysaccharide capsules, the determinant of serotype (or type), are heterogeneous in structure and amount and these differences affect rates of transmission. In this study, we examined the contribution of capsule and its variations to the maintenance of pneumococcal viability under starvation conditions. S. pneumoniae retained its ability to colonize infant mice even after incubation for 24hrs in PBS at 25°C. The expression of capsule by the cps locus prolonged survival under these and other nutrient-poor conditions. Analysis of capsule-switch constructs showed that strain-to-strain differences in survival were due to capsule type rather than genetic background. The addition of glucose was sufficient to rescue the survival defect of the capsule-deficient derivative demonstrating that in the absence of capsule survival depends upon nutrient availability. During starvation, there was a decrease in capsule size and amount of capsular polysaccharide that was dependent on bacterial viability and the presence of the cps locus. These observations suggest that pneumococci catabolize their own capsular polysaccharide using the genes involved in its biosynthesis to maintain viability when other carbon sources are unavailable. Our findings describe a new role of the pneumococcal capsule; the prolongation of viability under nutrient limiting conditions as would be encountered during periods when the organism is between hosts.
PMCID:5820961
PMID: 29311231
ISSN: 1098-5522
CID: 2906512

Platelet Transcriptome Profiling in HIV and ATP-Binding Cassette Subfamily C Member 4 (ABCC4) as a Mediator of Platelet Activity

Marcantoni, Emanuela; Allen, Nicole; Cambria, Matthew R; Dann, Rebecca; Cammer, Michael; Lhakhang, Tenzin; O'Brien, Meagan P; Kim, Benjamin; Worgall, Tilla; Heguy, Adriana; Tsirigos, Aristotelis; Berger, Jeffrey S
An unbiased platelet transcriptome profile identified ATP binding cassette subfamily C member 4 (ABCC4) as a novel mediator of platelet activity in virologically suppressed human immunodeficiency virus (HIV)-infected subjects on antiretroviral therapy. Using ex vivo and in vitro cellular and molecular assays we demonstrated that ABCC4 regulated platelet activation by altering granule release and cyclic nucleotide homeostasis through a cAMP-protein kinase A (PKA)-mediated mechanism. Platelet ABCC4 inhibition attenuated platelet activation and effector cell function by reducing the release of inflammatory mediators, such as sphingosine-1-phosphate. ABCC4 inhibition may represent a novel antithrombotic strategy in HIV-infected subjects on antiretroviral therapy.
PMCID:6058944
PMID: 30062189
ISSN: 2452-302x
CID: 3217022

Autophagy protein ATG16L1 prevents necroptosis in the intestinal epithelium

Matsuzawa-Ishimoto, Yu; Shono, Yusuke; Gomez, Luis E; Hubbard-Lucey, Vanessa M; Cammer, Michael; Neil, Jessica; Dewan, M Zahidunnabi; Lieberman, Sophia R; Lazrak, Amina; Marinis, Jill M; Beal, Allison; Harris, Philip A; Bertin, John; Liu, Chen; Ding, Yi; van den Brink, Marcel R M; Cadwell, Ken
A variant of the autophagy gene ATG16L1 is associated with Crohn's disease, an inflammatory bowel disease (IBD), and poor survival in allogeneic hematopoietic stem cell transplant recipients. We demonstrate that ATG16L1 in the intestinal epithelium is essential for preventing loss of Paneth cells and exaggerated cell death in animal models of virally triggered IBD and allogeneic hematopoietic stem cell transplantation. Intestinal organoids lacking ATG16L1 reproduced this loss in Paneth cells and displayed TNFalpha-mediated necroptosis, a form of programmed necrosis. This cytoprotective function of ATG16L1 was associated with the role of autophagy in promoting mitochondrial homeostasis. Finally, therapeutic blockade of necroptosis through TNFalpha or RIPK1 inhibition ameliorated disease in the virally triggered IBD model. These findings indicate that, in contrast to tumor cells in which autophagy promotes caspase-independent cell death, ATG16L1 maintains the intestinal barrier by inhibiting necroptosis in the epithelium.
PMCID:5716041
PMID: 29089374
ISSN: 1540-9538
CID: 2765882

Capsule Type and Amount Affect Shedding and Transmission of Streptococcus pneumoniae

Zafar, M Ammar; Hamaguchi, Shigeto; Zangari, Tonia; Cammer, Michael; Weiser, Jeffrey N
The capsular polysaccharide (CPS) of Streptococcus pneumoniae is characterized by its diversity, as it has over 95 known serotypes, and the variation in its thickness as it surrounds an organism. While within-host effects of CPS have been studied in detail, there is no information about its contribution to host-to-host transmission. In this study, we used an infant mouse model of intralitter transmission, together with isogenic capsule switch and cps promoter switch constructs, to explore the effects of CPS type and amount. The determining factor in the transmission rate in this model is the number of pneumococci shed in nasal secretions by colonized hosts. Two of seven capsule switch constructs showed reduced shedding. These constructs were unimpaired in colonization and expressed capsules similar in size to those of the wild-type strain. A cps promoter switch mutant expressing ~50% of wild-type amounts of CPS also displayed reduced shedding without a defect in colonization. Since shedding from the mucosal surface may require escape from mucus entrapment, a mucin-binding assay was used to compare capsule switch and cps promoter switch mutants. The CPS type or amount constructs that shed poorly were bound more robustly by immobilized mucin. These capsule switch and cps promoter switch constructs with increased mucin-binding affinity and reduced shedding also had lower rates of pup-to-pup transmission. Our results demonstrate that CPS type and amount affect transmission dynamics and may contribute to the marked differences in prevalence among pneumococcal types.IMPORTANCEStreptococcus pneumoniae, a leading cause of morbidity and mortality, is readily transmitted, especially among young children. Its structurally and antigenically diverse capsular polysaccharide is the target of currently licensed pneumococcal vaccines. Epidemiology studies show that only a subset of the >95 distinct serotypes are prevalent in the human population, suggesting that certain capsular polysaccharide types might be more likely to be transmitted within the community. Herein, we used an infant mouse model to show that both capsule type and amount are important determinants in the spread of pneumococci from host to host. Transmission rates correlate with those capsule types that are better at escaping mucus entrapment, a key step in exiting the host upper respiratory tract. Hence, our study provides a better mechanistic understanding of why certain pneumococcal serotypes are more common in the human population.
PMCID:5565965
PMID: 28830943
ISSN: 2150-7511
CID: 2676172

The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis

Hanna, Samer J; McCoy-Simandle, Kessler; Miskolci, Veronika; Guo, Peng; Cammer, Michael; Hodgson, Louis; Cox, Dianne
Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.
PMCID:5561213
PMID: 28819224
ISSN: 2045-2322
CID: 2669052

Gradients of the signaling lipid S1P in lymph nodes position natural killer cells and regulate their interferon-gamma response

Fang, Victoria; Chaluvadi, V Sai; Ramos-Perez, Willy D; Mendoza, Alejandra; Baeyens, Audrey; Rivera, Richard; Chun, Jerold; Cammer, Michael; Schwab, Susan R
The lymph node periphery is an important site for many immunological functions, from pathogen containment to the differentiation of helper T cells, yet the cues that position cells in this region are largely undefined. Here, through the use of a reporter for the signaling lipid S1P (sphingosine 1-phosphate), we found that cells sensed higher concentrations of S1P in the medullary cords than in the T cell zone and that the S1P transporter SPNS2 on lymphatic endothelial cells generated this gradient. Natural killer (NK) cells are located at the periphery of the lymph node, predominantly in the medulla, and we found that expression of SPNS2, expression of the S1P receptor S1PR5 on NK cells, and expression of the chemokine receptor CXCR4 were all required for NK cell localization during homeostasis and rapid production of interferon-gamma by NK cells after challenge. Our findings elucidate the spatial cues for NK cell organization and reveal a previously unknown role for S1P in positioning cells within the medulla.
PMCID:5675020
PMID: 27841869
ISSN: 1529-2916
CID: 2310882

Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability

Santos, Luís C; Blair, David A; Kumari, Sudha; Cammer, Michael; Iskratsch, Thomas; Herbin, Olivier; Alexandropoulos, Konstantina; Dustin, Michael L; Sheetz, Michael P
The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell-cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside-out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses.
PMCID:5121033
PMID: 27359298
ISSN: 1440-1711
CID: 3105992

S1P gradients position NK cells in lymph nodes and regulate the early IFN gamma response to infection [Meeting Abstract]

Fang, V; Ramos-Perez, WD; Chaluvadi, VS; Mendoza, A; Chun, J; Cammer, M; Schwab, SR
ISI:000383610402726
ISSN: 1521-4141
CID: 2283592

A map of the distribution of sphingosine 1-phosphate in the spleen

Ramos-Perez, Willy D; Fang, Victoria; Escalante-Alcalde, Diana; Cammer, Michael; Schwab, Susan R
Despite the importance of signaling lipids, many questions remain about their function because few tools are available for charting lipid gradients in vivo. Here we generated a sphingosine 1-phosphate (S1P) reporter mouse and used this mouse to define the distribution of S1P in the spleen. Unexpectedly, the presence of blood did not serve as a predictor of the concentration of signaling-available S1P. Large areas of the red pulp had low concentrations of S1P, while S1P was sensed by cells inside the white pulp near the marginal sinus. The lipid phosphate phosphatase LPP3 maintained low S1P concentrations in the spleen and enabled efficient shuttling of marginal zone B cells. The exquisitely tight regulation of S1P availability might explain how a single lipid can simultaneously orchestrate the movements of many cells of the immune system.
PMCID:4690472
PMID: 26502404
ISSN: 1529-2916
CID: 1817452

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells

Cespedes, Pablo F; Bueno, Susan M; Ramirez, Bruno A; Gomez, Roberto S; Riquelme, Sebastian A; Palavecino, Christian E; Mackern-Oberti, Juan Pablo; Mora, Jorge E; Depoil, David; Sacristan, Catarina; Cammer, Michael; Creneguy, Alison; Nguyen, Tuan H; Riedel, Claudia A; Dustin, Michael L; Kalergis, Alexis M
Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse- and human cell-based studies have shown that hRSV infection prevents naive T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4+ T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell-bilayer interface, suggesting that N protein interferes with pMHC-TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.
PMCID:4128097
PMID: 25056968
ISSN: 0027-8424
CID: 1076122