Eduardo Diez

Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila

In inbred mouse strains, permissiveness to intracellular replication of Legionella pneumophila is controlled by a single locus (Lgn1), which maps to a region within distal Chromosome 13 that contains multiple copies of the gene baculoviral IAP repeat–containing 1 (Birc1, also called Naip; refs. 1–3). Genomic BAC clones from the critical interval were transferred into transgenic mice to functionally complement the Lgn1-associated susceptibility of A/J mice to L. pneumophila. Here we report that two independent BAC clones that rescue susceptibility have an overlapping region of 56 kb in which the entire Lgn1 transcript must lie. The only known full-length transcript coded in this region is Birc1e (also called Naip5).

The neuronal apoptosis inhibitory protein (Naip) is expressed in macrophages and is modulated after phagocytosis and during intracellular infection with Legionella pneumophila.

Legionella pneumophila is an intracellular pathogen that causes Legionnaires’ disease in humans. Inbred mouse strains are uniformly resistant to L. pneumophila infection with the notable exception of A/J, where the chromosome 13 locus Lgn1 renders A/J macrophages permissive to L. pneumophila replication. The mouse Lgn1 region is syntenic with the spinal muscular atrophy (SMA) locus on human chromosome 5 and includes several copies of the neuronal apoptosis inhibitory protein (Naip) gene. We have analyzed a possible link among Lgn1, Naip, and macrophage function. RNA expression studies show that Naip (mostly copy 2) mRNA transcripts are expressed in macrophage-rich tissues, such as spleen, lung, and liver and are abundant in primary macrophages. Immunoblotting and immunoprecipitation analyses identify Naip protein expression in mouse macrophages and in macrophage cell lines RAW 264.7 and J774A. Interestingly, macrophages from permissive A/J mice express significantly less Naip protein than their nonpermissive C57BL/6J counterpart. Naip protein expression is increased after phagocytic events. Naip protein levels during infection with either virulent or avirulent strains of L. pneumophila increase during the first 6 h postinfection and remain elevated during the 48-h observation period. This enhanced expression is also observed in macrophages infected with Salmonella typhimurium. Likewise, an increase in Naip protein levels in macrophages is observed 24 h after phagocytosis of Latex beads. The cosegregation of Lgn1 and Naip together with the detected Naip protein expression in host macrophages as well as its modulation after phagocytic events and during intracellular infection make it an attractive candidate for the Lgn1 locus.

R-Spondin 2 signalling mediates susceptibility to fatal infectious diarrhoea

Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene (Rspo2) as a major determinant of susceptibility to C. rodentium infection. Robust induction of Rspo2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R spondins and Wnt signaling in susceptibility to infectious diarrhea and identify Rspo2 as a key molecular link between infection and intestinal homeostasis.

Endothelin-2-Mediated Protection of Mutant Photoreceptors in Inherited Photoreceptor Degeneration

Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2−/− mice carrying homozygous Pde6brd1 alleles or the Tg(RHO P347S) transgene. In the Edn2−/− background, PR survival increased 110% in Pde6brd1/rd1 mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6brd1/rd1; Edn2−/− mice. This finding, together with systemic abnormalities in Edn2−/− mice, suggested that the increased survival of mutant PRs in the Edn2−/− background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6brd1/rd1; Edn2−/− PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6brd1/rd1 retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2−/− retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

cDNA cloning and the 5'genomic organization of Naip2, a candidate gene for murine Legionella resistance.

NAIP (Neuronal Apoptosis Inhibitory Protein), cloned as a candidate gene for SMA (Spinal Muscular Atrophy; Morrison 1996), exists in multiple copies in the SMA region of Chr 5 (5q13; Roy et al. 1995). Deletions in the first two coding exons of the functional copy of NAIP are associated with approximately 66% of the type I SMA cases (Rodrigues et al. 1996; Velasco et al. 1996) and a lower percentage of the milder cases. The NAIP protein contains three baculovirus-inhibitor-of-apoptosis-protein-repeat (BIR) motifs (Roy et al. 1995) common to a family of apoptosis inhibitory proteins (Liston et al. 1996), and is present in motor neurons and other neuronal populations affected in type I SMA (Xu et al. 1997a). Furthermore, NAIP has been shown to inhibit apoptosis both in vitro (Liston et al. 1996) and in vivo (Xu et al. 1997b). The neuroprotective nature of NAIP, its expression pattern, as well as the association of NAIP deletions with the severe cases of SMA, make NAIP a strong candidate as a phenotypic modifier of SMA. Recently we reported the cloning of multiple homologs of NAIP in the mouse, designated Naip1–6. Three of these loci (Naip1–3) contain the 58UTR cloned from brain RNA, and necessary for translation (Yaraghi et al. 1998). These three loci not only show sequence differences at the cDNA level, but also a high degree of divergence both in size and sequence of introns. This observation suggests that, in contrast to the human case with one functional NAIP gene and multiple unprocessed truncated and deleted pseudogenes, the mouse possesses different, full-length Naip loci representing a family of genes. One possible reason for this genomic redundancy may be tissue-specific expression of each member of the gene family. Recent studies have localized the multiple Naip loci within the Lgn1critical interval of mouse Chr 13 (region D1–D3; Scharf et al. 1996; Diez et al. 1997). The Lgn1 locus is responsible for modulating the intracellular replication and pathogenicity of the bacterium Legionella pneumophila, the causative agent of Legionnaires’ disease (Beckers et al. 1995; Dietrich et al. 1995). An important aspect of the pathogenicity of Legionellais the infection of macrophages with the bacterium (Ciancotto et al. 1989). Although primary macrophages and macrophage cell lines from human and guinea pig sustain infection with Legionella(Marra and Shuman 1992), primary macrophages from most inbred mouse strains are nonpermissive to the intracellular replication of the bacterium. An exception to murineLegionella resistance is the A/J strain, the inflammatory macrophages of which are permissive for Legionella replication (Yamamoto et al. 1988). The overlap between the Naip1–6-containing region of Chr 13 and the Lgn1critical interval points toNaip as a candidate gene for Legionellaresistance. Supporting this role forNaip is the recent report suggesting that cell cytotoxicity by Legionella pneumophilaoccurs at least in part through apoptosis (Muller et al. 1996). We postulate that A/J macrophages deficient in the critical Naip locus may become apoptotic upporting intracellular bacterial proliferation. In resistant mouse strains, however, the presence of Naipmay inhibit apoptosis of the macrophages, allowing them to phagocytose the bacteria and inhibiting their replication. In order to assess the possibility of tissue-specific expression of each one of theNaip1, 2,and3 loci, we carried out an RT-PCR analysis of mouse C57BL/6J brain and spleen RNA. Primer pairs utilized for this analysis were designed based on the sequence divergence seen within exon 1 and 4 of Naip1–3(Yaraghi et al. 1998) and were specific to each one of these three loci. As shown in Fig. 1, 60 cycles of amplification were not sufficient to detect either Naip1 or Naip3 transcript in spleen. Thus, the levels of Naip1 and Naip3 transcripts appear negligible in the spleen. We cannot, however, formally exclude the existence of Naip1 and Naip3 isoforms, which would not be amplified under the conditions of the RT-PR assay. Our results thus identify Naip2 as a spleen-transcribed and Naip1 as a brain-transcribed locus. A further characterization of the Naip2 locus was undertaken. Sequence analysis of cDNA clones from a mouse C57BL/6J spleen cDNA library (Yaraghi et al. 1998) identified a 4.9-kb Naip2 cDNA clone (ms2), which contained the complete coding region, the 58UTR, and parts of the 3 8UTR (Naip2 cDNA, GenBank accession number AF102871). Naip2 encodes a protein of 1447 amino acids with a predicted size of 164 kDa. The Naip2 coding region revealed 90.4% nucleotide sequence homology to Naip1and 77% to human NAIP. The Naip2 transcript contains an additional exon (exon 9a) not seen in Naip1.With primers specific to this exon, PCR analysis mapped the 132-bp exon only to 129/ SvJ genomic BAC clones harboring Naip2 (Yaraghi et al. 1998), suggesting its exclusive presence in this locus (data not shown). Exon 9a does not cause an interruption of the open reading frame, and its predicted amino acid sequence contains no known motifs. A high degree of divergence between the Naip1 andNaip2 transcripts is seen within the first 140 bp of exon 2 (25%). In both cDNAs, the predicted amino acid sequence of this region does not contain any known motifs and lies upstream of the first BIR domain (Roy et al. 1995). PCR analysis of genomic BAC clones harboring all six differentNaip loci using primers specific to the first 140 bp ofNaip2exon 2 revealed the exclusive presence of this sequence inNaip2 (data not shown). BothNaip1andNaip2show similar levels of amino acid identity to human NAIP (68.4% and 68.9% respectively), while sharing an 86.6% identity (excluding exon 9a). The three BIR domains encoded by exons 2–8 are highly Correspondence to: A. MacKenzie at Molecular Genetics, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario K1H 8L1, Canada.

The Cri1 locus is the common genetic cause of susceptibility to Citrobacter rodentium infection in C3H and FVB mouse strains.

Citrobacter rodentium is a natural pathogen of mice that causes intestinal hyperplasia and colitis. Resistant strains such as C57BL/6J (B6) experience a self-limiting disease that peaks between one and two weeks post infection, followed by a clearing of the infection and complete recovery. However, the inbred mouse strains C3H/HeJ (C3), C3H/HeOuJ (C3Ou) and FVB/N (FVB) are highly susceptible to C. rodentium infection and develop more severe symptoms of disease leading to high rates of mortality during infection. We have recently demonstrated through a systematic genetics approach that a single locus on proximal chromosome 15 is responsible for the susceptibility of both C3 and C3Ou mice to C. rodentium infection. We have named the locus Citrobacter rodentium infection 1 (Cri1). Here we show that Cri1 also controls susceptibility to C. rodentium in FVB mice, using a targeted method of genotyping to stratify (B6 x FVB)F2 mice according to their genotype at Cri1. Mice that inherit two copies of the resistant B6 allele have 97% cumulative survival at day 30 post-infection, whereas those that inherit one or two copies of Cri1 from the FVB parent have significantly lower rates of survival (35% and 42%, respectively). These results provide evidence for a common genetic cause of fatal infectious colitis in C3, C3Ou and FVB mice following infection with Citrobacter rodentium.

Positional cloning of a quantitative trait locus contributing to pain sensitivity: possible mediation by Tyrp1

To identify novel pain‐relevant genes, a set of 35 recombinant congenic strains derived from the sensitive C57BL/6 and resistant A/J strains were tested for their sensitivity to noxious heat on the radiant heat paw‐withdrawal test. Nine strains were found to display differential sensitivity, and the two most extreme responders were used to generate independent secondary crosses for quantitative trait locus (QTL) mapping. From these genetic analyses, a QTL, which we call Tpnr5, was mapped to a 14‐Mb interval of mouse chromosome 4 containing 39 genes. In addition to the paw‐withdrawal test phenotype, Tpnr5 may be relevant to mechanical and inflammatory nociception. A series of strategies – including in silico analyses, reverse transcriptase polymerase chain reaction (RT‐PCR) in multiple tissues and exonic DNA sequencing – were used to generate a list of six prioritized candidate genes. One of these, tyrosinase‐related protein 1 (Tyrp1), displayed enriched expression in the dorsal root ganglia, an inactivating (C110Y) mutation in the resistant A/J strain, and a null mutant found to be more resistant to thermal nociception compared to its wild‐type counterpart. Although other genes cannot be definitively ruled out, existing data are supportive of the candidacy of Tyrp1 as representing the Tpnr5 QTL. Tyrosinase‐related protein 1 is the rate‐limiting enzyme in the production of eumelanin, and possible relationships between eumelanin‐expressing cells and thermal nociception are discussed. The positional cloning of Tpnr5 is also considered in light of the heuristic value but continuing challenges of QTL mapping in the mouse.

Genetic control of high density lipoprotein-cholesterol in AcB/BcA recombinant congenic strains of mice

Epidemiological studies show that high HDL-cholesterol (HDLc) decreases the risk of cardiovascular disease. To map genes controlling lipid metabolism, particularly HDLc levels, we screened the plasma lipids of 36 AcB/BcA RC mouse strains subjected to either a normal or a high-fat/cholesterol diet. Strains BcA68 and AcB65 showed deviant HDLc plasma levels compared with the parental A/J and C57BL/6J strains; they were thus selected to generate informative F2 crosses. Linkage analyses in the AcB65 strain identified a locus on chromosome 4 (Hdlq78) responsible for high post-high fat diet HDLc levels. This locus has been previously associated at genome-wide significance to two regions in the human genome. A second linkage analysis in strain BcA68 identified linkage in the vicinity of a gene cluster known to control HDLc levels. Sequence analysis of these candidates identified a de novo, loss-of-function mutation in the ApoA1 gene of BcA68 that prematurely truncates the ApoA1 protein. The possibility of dissecting the specific effects of this new ApoA1 deficiency in the context of isogenic controls makes the BcA68 mouse a valuable new tool.