Glycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to Francisella tularensis.

Francisella tularensis is the causative agent of tularemia, a category A bioterrorism agent. The lipopolysaccharide (LPS) O antigen (OAg) of F. tularensis has been considered for use in a glycoconjugate vaccine, but conjugate vaccines tested so far have failed to confer protection necessary against aerosolized pulmonary bacterial challenge. When F. tularensis OAg was purified under standard conditions, the antigen had a small molecular size [25 kDa, low molecular weight (LMW)]. Using milder extraction conditions, we found the native OAg had a larger molecular size [80 kDa, high molecular weight (HMW)], and in a mouse model of tularemia, a glycoconjugate vaccine made with the HMW polysaccharide coupled to tetanus toxoid (HMW-TT) conferred better protection against intranasal challenge than a conjugate made with the LMW polysaccharide (LMW-TT). To further investigate the role of OAg size in protection, we created an F. tularensis live vaccine strain (LVS) mutant with a significantly increased OAg size [220 kDa, very high molecular weight (VHMW)] by expressing in F. tularensis a heterologous chain-length regulator gene (wzz) from the related species Francisella novicida Immunization with VHMW-TT provided markedly increased protection over that obtained with TT glycoconjugates made using smaller OAgs. We found that protective antibodies recognize a length-dependent epitope better expressed on HMW and VHMW antigens, which bind with higher affinity to the organism.

Symbionts exploit complex signaling to educate the immune system.

The mammalian immune system is tolerized to trillions of microbes residing on bodily surfaces and can discriminate between symbionts and pathogens despite their having related microbial structures. Mechanisms of innate immune activation and the subsequent signaling pathways used by symbionts to communicate with the adaptive immune system are poorly understood. Polysaccharide A (PSA) of Bacteroides fragilis is the model symbiotic immunomodulatory molecule. Here we demonstrate that PSA-dependent immunomodulation requires the Toll-like receptor (TLR) 2/1 heterodimer in cooperation with Dectin-1 to initiate signaling by the downstream phosphoinositide 3-kinase (PI3K) pathway, with consequent CREB-dependent transcription of antiinflammatory genes, including antigen presentation and cosignaling molecules. High-resolution LC-MS/MS analysis of PSA identified a previously unknown small molecular-weight, covalently attached bacterial outer membrane-associated lipid that is required for activation of antigen-presenting cells. This archetypical commensal microbial molecule initiates a complex collaborative integration of Toll-like receptor and C-type lectin-like receptor signaling mechanisms culminating in the activation of the antiinflammatory arm of the PI3K pathway that serves to educate CD4+ Tregs to produce the immunomodulatory cytokine IL-10. Immunomodulation is a key function of the microbiome and is a focal point for developing new therapeutic agents.

Phenotypic, morphological, and functional heterogeneity of splenic immature myeloid cells in the host response to tularemia.

Recent studies have linked accumulation of the Gr-1⁺ CD11b⁺ cell phenotype with functional immunosuppression in diverse pathological conditions, including bacterial and parasitic infections and cancer. Gr-1⁺ CD11b⁺ cells were the largest population of cells present in the spleens of mice infected with sublethal doses of the Francisella tularensis live vaccine strain (LVS). In contrast, the number of T cells present in the spleens of these mice did not increase during early infection. There was a significant delay in the kinetics of accumulation of Gr-1⁺ CD11b⁺ cells in the spleens of B-cell-deficient mice, indicating that B cells play a role in recruitment and maintenance of this population in the spleens of mice infected with F. tularensis. The splenic Gr-1⁺ CD11b⁺ cells in tularemia were a heterogeneous population that could be further subdivided into monocytic (mononuclear) and granulocytic (polymorphonuclear) cells using the Ly6C and Ly6G markers and differentiated into antigen-presenting cells following ex vivo culture. Monocytic, CD11b⁺ Ly6C(hi) Ly6G⁻ cells but not granulocytic, CD11b⁺ Ly6C(int) Ly6G⁺ cells purified from the spleens of mice infected with F. tularensis suppressed polyclonal T-cell proliferation via a nitric oxide-dependent pathway. Although the monocytic, CD11b⁺ Ly6C(hi) Ly6G⁻ cells were able to suppress the proliferation of T cells, the large presence of Gr-1⁺ CD11b⁺ cells in mice that survived F. tularensis infection also suggests a potential role for these cells in the protective host response to tularemia.

Evidence that two ATP-dependent (Lon) proteases in Borrelia burgdorferi serve different functions.

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH(2)-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host.

The smpB-ssrA mutant of Yersinia pestis functions as a live attenuated vaccine to protect mice against pulmonary plague infection.

The bacterial SmpB-SsrA system is a highly conserved translational quality control mechanism that helps maintain the translational machinery at full capacity. Here we present evidence to demonstrate that the smpB-ssrA genes are required for pathogenesis of Yersinia pestis, the causative agent of plague. We found that disruption of the smpB-ssrA genes leads to reduction in secretion of the type III secretion-related proteins YopB, YopD, and LcrV, which are essential for virulence. Consistent with these observations, the smpB-ssrA mutant of Y. pestis was severely attenuated in a mouse model of infection via both the intranasal and intravenous routes. Most significantly, intranasal vaccination of mice with the smpB-ssrA mutant strain of Y. pestis induced a strong antibody response. The vaccinated animals were well protected against subsequent lethal intranasal challenges with virulent Y. pestis. Taken together, our results indicate that the smpB-ssrA mutant of Y. pestis possesses the desired qualities for a live attenuated cell-based vaccine against pneumonic plague.

Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes.

The histone acetyltransferases p300 and cAMP-responsive element-binding protein-binding protein (CBP) are required for the execution of critical biological functions such as proliferation, differentiation, and apoptosis. Both proteins are believed to regulate the activity of a large number of general and cell-specific transcription factors. Here we demonstrate a dramatic decrease in the total cellular levels of p300 and CBP with increasing population doublings of human normal melanocytes. We show that one consequence of p300 depletion is transcriptional down-regulation of the cyclin E gene, caused by deacetylation of histones at its promoter. The cyclin E promoter was activated by p300 and the histone deacetylase inhibitor trichostatin A. Conversely, the cyclin E promoter was repressed by wild-type Retinoblastoma tumor suppressor p105 protein (pRB) and by a dominant negative p300 mutant (DN p300) that lacks histone acetyltransferase activity. We also provide evidence of the alternative recruitment of p300 and histone deacetylase 1 to the cyclin E promoter in proliferating and senescent melanocytes, respectively. The biological significance of these results was established by showing that block of p300 activity by overexpression of DN p300 or by Lys-CoA, a specific chemical inhibitor of p300, resulted in growth inhibition, down-regulation of cyclin E, and activation of the senescence-associated beta-galactosidase marker in human melanocytes and melanoma cells. Together, these results provide evidence for the essential role of p300 in the regulation of proliferation and senescence in cells from melanocytic origin.

Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19.

UNLABELLED: Differences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background. IMPORTANCE: Francisella tularensis is a highly pathogenic bacterium with a very low infectious dose in humans. Some mechanisms of bacterial virulence have been elucidated, but the host genetic factors that contribute to host resistance or susceptibility are largely unknown. In this work, we have undertaken a genetic approach to assess what these factors are in mice. Analyzing early interactions of macrophages with the bacteria as well as data on overall susceptibility to infection revealed a locus on chromosome 19 that is associated with both phenotypes. In addition, our work revealed differences in the early macrophage response between macrophages with different genetic backgrounds. Overall, this work suggests some intriguing links between in vitro and in vivo infection models and should aid in further elucidating the genetic circuits behind the host response to Francisella tularensis infection.

The atypical lipopolysaccharide of Francisella.

Bacterial lipopolysaccharides (LPSs) are ubiquitous molecules that are prominent components of the outer membranes of most gram-negative bacteria. Genetic and structural characterizations of Francisella LPS have revealed substantial differences when compared to more commonly studied LPSs of the Enterobacteriaceae. This review discusses both the general characteristics and the unusual features of Francisella LPS.

Kdo hydrolase is required for Francisella tularensis virulence and evasion of TLR2-mediated innate immunity.

UNLABELLED: The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD(50)) 2 log(10) units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1ß in a TLR2-dependent manner. In addition, TLR2(-/-) mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine. IMPORTANCE: The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification-Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal--has recently been reported in F. tularensis. We found that the kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.

Trans-translation: the tmRNA-mediated surveillance mechanism for ribosome rescue, directed protein degradation, and nonstop mRNA decay.

The accurate flow of genetic information from DNA to RNA to protein is essential for all living organisms. An astonishing array of quality-assurance mechanisms have evolved to ensure that high degree of fidelity is maintained at every stage of this process. One of the most fascinating quality-control mechanisms involves tmRNA, also known as SsrA or 10Sa RNA. tmRNA is a versatile and highly conserved bacterial molecule endowed with the combined structural and functional properties of both a tRNA and a mRNA. The tmRNA system orchestrates three key biological functions: (1) recognition and rescue of ribosomes stalled on aberrant mRNAs, (2) disposal of the causative defective mRNAs, and (3) addition of a degradation tag to ribosome-associated protein fragments for directed proteolysis. Although not essential in Escherichia coli, tmRNA activity is essential for bacterial survival under adverse conditions and for virulence in some, and perhaps all, pathogenic bacteria. Recent evidence suggests that in addition to its quality-control function the tmRNA system might also play a key regulatory role in certain physiological pathways. This review will focus on recent advances in our understanding of the structural properties, mechanistic details, and physiological significance of this unique RNA and its principal protein partners.

A Role for the SmpB-SsrA system in Yersinia pseudotuberculosis pathogenesis.

Yersinia utilizes a sophisticated type III secretion system to enhance its chances of survival and to overcome the host immune system. SmpB (small protein B) and SsrA (small stable RNA A) are components of a unique bacterial translational control system that help maintain the bacterial translational machinery in a fully operational state. We have found that loss of the SmpB-SsrA function causes acute defects in the ability of Yersinia pseudotuberculosis to survive in hostile environments. Most significantly, we show that mutations in smpB-ssrA genes render the bacterium avirulent and unable to cause mortality in mice. Consistent with these observations, we show that the mutant strain is unable to proliferate in macrophages and exhibits delayed Yop-mediated host cell cytotoxicity. Correspondingly, we demonstrate that the smpB-ssrA mutant suffers severe deficiencies in expression and secretion of Yersinia virulence effector proteins, and that this defect is at the level of transcription. Of further interest is the finding that the SmpB-SsrA system might play a similar role in the related type III secretion system that governs flagella assembly and bacterial motility. These findings highlight the significance of the SmpB-SsrA system in bacterial pathogenesis, survival under adverse environmental conditions, and motility.