New insights into the crosstalk between Shigella and T lymphocytes

International audienceSubversion of host immune responses is the key infection strategy employed by most, if not all, human pathogens. Modulation of the host innate response by pathogens has been vastly documented. Yet, especially for bacterial infections, it was only recently that cells of the adaptive immune response were recognized as targets of bacterial weapons such as the type III secretion system (T3SS) and its effector proteins. In this review, we focus on the recent advances made in the understanding of how the enteroinvasive bacterium Shigella flexneri interferes with the host adaptive response by targeting T lymphocytes, especially their migration capacities

Shigella impairs T lymphocyte dynamics in vivo

International audienceThe Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium-T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri, via its type III secretion system, impairs the migration pattern of CD4(+) T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4(+) T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4(+) T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses

The Spl Serine Proteases Modulate Staphylococcus aureus Protein Production and Virulence in a Rabbit Model of Pneumonia

ABSTRACT The Spl proteases are a group of six serine proteases that are encoded on the νSaβ pathogenicity island and are unique to Staphylococcus aureus. Despite their interesting biochemistry, their biological substrates and functions in virulence have been difficult to elucidate. We found that an spl operon mutant of the community-associated methicillin-resistant S. aureus USA300 strain LAC induced localized lung damage in a rabbit model of pneumonia, characterized by bronchopneumonia observed histologically. Disease in the mutant-infected rabbits was restricted in distribution compared to that in wild-type USA300-infected rabbits. We also found that SplA is able to cleave the mucin 16 glycoprotein from the surface of the CalU-3 lung cell line, suggesting a possible mechanism for wild-type USA300 spreading pneumonia to both lungs. Investigation of the secreted and surface proteomes of wild-type USA300 and the spl mutant revealed multiple alterations in metabolic proteins and virulence factors. This study demonstrates that the Spls modulate S. aureus physiology and virulence, identifies a human target of SplA, and suggests potential S. aureus targets of the Spl proteases. IMPORTANCE Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade both staphylococcal and human proteins

The Shigella flexneri Type Three Secretion System Effector IpgD Inhibits T Cell Migration by Manipulating Host Phosphoinositide Metabolism.

International audienceShigella, the Gram-negative enteroinvasive bacterium that causes shigellosis, relies on its type III secretion system (TTSS) and injected effectors to modulate host cell functions. However, consequences of the interaction between Shigella and lymphocytes have not been investigated. We show that Shigella invades activated human CD4(+) T lymphocytes. Invasion requires a functional TTSS and results in inhibition of chemokine-induced T cell migration, an effect mediated by the TTSS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Upon IpgD-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), the pool of PIP(2) at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus, likely affecting the formation of the polarized edge required for cell migration. These results reveal a bacterial TTSS effector-mediated strategy to impair T cell function

Th17 cells are the dominant T cell subtype primed by Shigella flexneri mediating protective immunity

International audienceThe T cell response to Shigella, the causative agent of bacillary dysentery, remains poorly understood. Using a murine model of infection, we report that Shigella flexneri primes predominately IL-17A- and IL-22-producing Th17 cells. Shigella-specific Th1 cells are only significantly induced on secondary infection, whereas specific Th2 and CD8(+) T cells are undetectable. Apart from Th17 cells that are primed in a MHC class II- and IL-6-dependent, but IL12/23p40-independent manner, we identified gammadelta T cells as an additional but minor source of IL-17A. Priming of IL-17A(+) gammadelta T cells is dependent on IL12/23p40, but independent of MHC-class II and IL-6. Th17 cells have emerged as important players in inflammatory, autoimmune, and infectious diseases. Among the yet unresolved questions is their role in long-term immunity to pathogens. In this study, we show that the elicited S. flexneri-specific Th17 pool gives rise to an enhanced recall response up to 12 mo after priming, suggesting the presence of a long-term memory state. The clearance of primary infection is impaired in the absence of T cells, but independently of IL-17A. However, after reinfection, IL-17A produced by S. flexneri-specific Th17 cells becomes important to ultimately restrict bacterial growth. These findings bring new insights into the adaptive immune response to Shigella infection and highlight the importance of pathogen-specific Th17 cell immunity for secondary immune protection

Functional Analysis of the Gonococcal Genetic Island of <i>Neisseria gonorrhoeae</i>

<div><p><i>Neisseria gonorrhoeae</i> is an obligate human pathogen that is responsible for the sexually-transmitted disease gonorrhea. <i>N. gonorrhoeae</i> encodes a T4SS within the Gonococcal Genetic Island (GGI), which secretes ssDNA directly into the external milieu. Type IV secretion systems (T4SSs) play a role in horizontal gene transfer and delivery of effector molecules into target cells. We demonstrate that GGI-like T4SSs are present in other β-proteobacteria, as well as in α- and γ-proteobacteria. Sequence comparison of GGI-like T4SSs reveals that the GGI-like T4SSs form a highly conserved unit that can be found located both on chromosomes and on plasmids. To better understand the mechanism of DNA secretion by <i>N. gonorrhoeae</i>, we performed mutagenesis of all genes encoded within the GGI, and studied the effects of these mutations on DNA secretion. We show that genes required for DNA secretion are encoded within the <i>yaa-atlA</i> and <i>parA-parB</i> regions, while genes encoded in the <i>yfeB-exp1</i> region could be deleted without any effect on DNA secretion. Genes essential for DNA secretion are encoded within at least four different operons.</p></div

Characterization of the <i>yaf-yaa</i> region.

<p>A) Fluorometric detection of secreted DNA of mutants created in the <i>yaf-yaa</i> region Gonococcal strains were repeatedly diluted in liquid culture. Cell-free culture supernatants were collected, and DNA was detected with the fluorescent DNA-binding dye PicoGreen and normalized to the increase in the OD₆₀₀. MS11 was used as the wild-type (WT) strain and ND500 (MS11ΔGGI) was used for the background value. <i>yaf^#</i> indicates the TB001 strain which contains a markerless in-frame deletion of <i>yaf</i>. <i>yaa*</i> indicates the TB002 strain in which the <i>yaa</i> gene is disrupted by insertion of an <i>ermC</i> containing plasmid via insertion duplication mutagenesis. <i>yaa^†</i> indicates the KL505 strain in which a 4 bp insertion creates a frame shift in <i>yaa</i>, and <i>yaa^†::yaa</i> indicates the TB003 strain in which the 4 bp insertion is removed via homologous recombination with WT <i>yaa</i>. B) Quantitative gene expression levels of the <i>traI</i>, <i>traD</i>, <i>ltgX</i>, <i>traH</i> and <i>parA</i> genes of non-piliated <i>N. gonorrhoeae</i> strains were determined by qRT-PCR. The graph shows the mRNA levels as comparative gene expression after normalizing each gene to <i>secY</i>. Values depict means ± standard deviation of at least six biological replicates. § indicates a Student's T-test P-value≤0.05 compared to wild-type; ‡, indicates no statistical difference compared to WT.</p

Schematic representation of the genetic map of the GGI of <i>N. gonorrhoeae</i>.

<p>Genes that have been previously characterized are colored. Red and orange indicate genes in which mutations resulted in a decrease in DNA secretion. Green indicates genes in which mutations had no effect on DNA secretion. For the genes colored in red, secretion could be restored by complementation. For the genes colored in orange no complementation was performed. The dot indicates the origin of transfer (<i>oriT</i>). difA and difB indicate the respective dif sites flanking the GGI.</p

Characterization of the <i>ltgX-atlA</i> region.

<p>A) Fluorometric detection of secreted DNA of mutants created in the <i>ltgX-atlA</i> region was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109613#pone-0109613-g002" target="_blank">Figure 2</a>. # indicates a strain which contains a markerless in-frame deletion of the respective gene. * indicates a strain in which the respective gene is disrupted by insertion of an <i>ermC</i> containing plasmid via insertion duplication mutagenesis. $ indicates a strain in which the gene is disrupted by replacement with the <i>ermC</i> cassette via double homologous recombination. <i>yag^#</i>::<i>yag⁺</i> indicates the complementation mutant of the in-frame deletion of <i>yag</i> by expressing <i>yag</i> from the complementation site between the <i>aspC</i> and <i>lctP</i> genes. <i>ych^&</i> indicates the strain in which <i>ych</i> is disrupted by a transposon insertion. § indicates a Student's T-test P-value≤0.05 compared to wild-type; ‡, indicates no statistical difference compared to WT.</p