Tara D. Wehrly

Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication

Intracellular bacterial pathogens evade the bactericidal functions of mammalian cells by physical escape from their phagosome and replication into the cytoplasm or through the modulation of phagosome maturation and biogenesis of a membrane-bound replicative organelle. Here, we detail in murine primary macrophages the intracellular life cycle of Francisella tularensis, a highly infectious bacterium that survives and replicates within mammalian cells. After transient interactions with the endocytic pathway, bacteria escaped from their phagosome by 1 h after infection and underwent replication in the cytoplasm from 4 to 20 h after infection. Unexpectedly, the majority of bacteria were subsequently found to be enclosed within large, juxtanuclear, LAMP-1-positive vacuoles called Francisella-containing vacuoles (FCVs). FCV formation required intracytoplasmic replication of bacteria. Using electron and fluorescence microscopy, we observed that the FCVs contained morphologically intact bacteria, despite fusing with lysosomes. FCVs are multimembranous structures that accumulate monodansylcadaverine and display the autophagy-specific protein LC3 on their membrane. Formation of FCVs was significantly inhibited by 3-methyladenine, confirming a role for the autophagic pathway in the biogenesis of these organelles. Taken together, our results demonstrate that, via autophagy, F. tularensis reenters the endocytic pathway after cytoplasmic replication, a process thus far undescribed for intracellular pathogens.

Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule

The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments. V(D)J recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking recombination signal sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction. The conserved heptamer and nonamer sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting, specifies that V(D)J recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS. Vβ segments are appended to DJβ rearrangements, with little or no direct Vβ to Jβ joining, despite 12/23 compatibility of Vβ 23-RSSs and Jβ12-RSSs. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)β locus containing only one Dβ (Dβ1) gene segment and one Jβ (Jβ1) gene cluster to show that the 5′ Dβ1 12-RSS, but not the Jβ1 12-RSSs, targets rearrangement of a diverse Vβ repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J recombination has important implications for the regulation of variable region gene assembly and repertoire development.

Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile.

Cutting Edge: Targeting of Vβ to Dβ Rearrangement by RSSs Can Be Mediated by the V(D)J Recombinase in the Absence of Additional Lymphoid-Specific Factors

Assembly of TCRβ variable region genes is ordered during thymocyte development with Dβ to Jβ rearrangement preceding Vβ to DJβ rearrangement. The 5′Dβ 12-RSS is required to precisely and efficiently target Vβ rearrangement beyond simply enforcing the 12/23 rule. By prohibiting direct Vβ to Jβ rearrangement, this restriction ensures Dβ gene segment use in the assembly of essentially all TCRβ variable region genes. In this study, we show that rearrangement of Vβ 23-RSSs is significantly biased to the Dβ 12-RSS over Jβ 12-RSSs on extrachromosomal recombination substrates in nonlymphoid cells that express the recombinase-activating gene-1/2 proteins. These findings demonstrate that targeting of Vβ to Dβ rearrangement can be enforced by the V(D)J recombinase in the absence of lymphoid-specific factors other than the recombinase-activating gene-1/2 proteins.

Acid Phosphatases Do Not Contribute to the Pathogenesis of Type A Francisella tularensis

ABSTRACT The intracellular pathogen Francisella tularensis is the causative agent of tularemia, a zoonosis that can affect humans with potentially lethal consequences. Essential to Francisella virulence is its ability to survive and proliferate within phagocytes through phagosomal escape and cytosolic replication. Francisella spp. encode a variety of acid phosphatases, whose roles in phagosomal escape and virulence have been documented yet remain controversial. Here we have examined in the highly virulent (type A) F. tularensis strain Schu S4 the pathogenic roles of three distinct acid phosphatases, AcpA, AcpB, and AcpC, that are most conserved between Francisella subspecies. Neither the deletion of acpA nor the combination of acpA, acpB, and acpC deletions affected the phagosomal escape or cytosolic growth of Schu S4 in murine and human macrophages, despite decreases in acid phosphatase activities by as much as 95%. Furthermore, none of these mutants were affected in their ability to cause lethality in mice upon intranasal inoculation. Hence, the acid phosphatases AcpA, AcpB, and AcpC do not contribute to intracellular pathogenesis and do not play a major role in the virulence of type A Francisella strains.

T cell receptor CDR3 loop length repertoire is determined primarily by features of the V(D)J recombination reaction.

The third complementarity‐determining region (CDR) of the TCR α and β chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic‐peptide–MHC complexes by the TCR. The TCRβ chain CDR3 loop is encoded by the Dβ gene segment and flanking portions of the Vβ and Jβ gene segments. The joining of these gene segments is imprecise, leading to significant variability in the TCRβ chain CDR3 loop length and amino acid composition. In marked contrast to other pairing antigen‐receptor chains, the TCR β and α chain CDR3 loop size distributions are relatively narrow and closely matched. Thus, pairing of TCR α and β chains with relatively similar CDR3 loop sizes may be important for generating a functional repertoire of α β TCR. Here we show that the TCRβ chain CDR3 loop size distribution is minimally impacted by TCRβ chain or α β TCR selection during thymocyte development. Rather, this distribution is determined primarily at the level of variable‐region gene assembly, and is critically dependent on unique features of the V(D)J recombination reaction that ensure Dβ gene segment utilization.

The B12/23 Restriction Is Critically Dependent on Recombination Signal Nonamer and Spacer Sequences

Ag receptor variable region gene assembly is initiated through the formation of a synaptic complex which minimally includes the recombination-activating gene (RAG) 1/2 proteins and a pair of recombination signals (RSs) flanking the recombining gene segments. RSs are composed of conserved heptamer and nonamer sequences flanking relatively nonconserved spacers of 12 or 23 bp. RSs regulate variable region gene assembly within the context of the 12/23 rule which mandates that recombination only occurs between RSs of dissimilar spacer length. RSs can exert additional constraints on variable region gene assembly beyond imposing spacer length requirements. At a minimum this restriction, termed B12/23, is imposed on the Vβ to DJβ rearrangement step by the 5′ Dβ RS and is enforced at or before the DNA cleavage step of the V(D)J recombination reaction. In this study, the components of the 5′ Dβ RS required for enforcing the B12/23 rule are assessed on chromosomal substrates in vivo in the context of normal murine thymocyte development and on extrachromosomal substrates induced to undergo recombination in nonlymphoid cell lines. These analyses reveal that the integrity of the nonamer sequence as well as the highly conserved spacer nucleotides of the 5′ Dβ1 RS are critical for enforcing the B12/23 restriction. These findings have important implications for understanding the B12/23 restriction and the manner in which RS synaptic complexes are assembled in vivo.

Restrictions limiting the generation of DNA double strand breaks during chromosomal V(D)J recombination

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.

Mitochondrial ROS potentiates indirect activation of the AIM2 inflammasome

Activation of the inflammasome is important for the detection and clearance of cytosolic pathogens. In contrast to avirulent Francisella novicida (Fn), infection with virulent Francisella tularensis ssp tularensis does not trigger activation of the host AIM2 inflammasome. Here we show that differential activation of AIM2 following Francisella infection is due to sensitivity of each isolate to reactive oxygen species (ROS). ROS present at the outset of Fn infection contributes to activation of the AIM2 inflammasome, independent of NLRP3 and NADPH oxidase. Rather, mitochondrial ROS (mROS) is critical for Fn stimulation of the inflammasome. This study represents the first demonstration of the importance of mROS in the activation of the AIM2 inflammasome by bacteria. Our results also demonstrate that bacterial resistance to mROS is a mechanism of virulence for early evasion of detection by the host.

Virulent Francisella tularensis destabilize host mRNA to rapidly suppress inflammation.

Highly virulent bacterial pathogens have evolved rapid means to suppress host inflammatory responses by unknown mechanisms. Here, we use virulent Francisella tularensis, the cause of lethal tularemia in humans, as a model to elucidate these mechanisms. We show that following infection of murine macrophages F. tularensis rapidly and selectively destabilizes mRNA containing adenylate-uridylate-rich elements that encode for cytokines and chemokines important in controlling bacterial infection. Degradation of host mRNA encoding interleukin (IL)-1β, IL-6 and CXCL1 did not require viable bacteria or de novo protein synthesis, but did require escape of intracellular organisms from endocytic vesicles into the host cytosol. The specific targeting of host mRNA encoding inflammatory cytokines and chemokines for decay by a bacterial pathogen has not been previously reported. Thus, our findings represent a novel strategy by which a highly virulent pathogen modulates host inflammatory responses critical to the evasion of innate immunity.