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Originally published online as doi:10.1189/jlb.1004616 on January 14, 2005

Published online before print January 14, 2005
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(Journal of Leukocyte Biology. 2005;77:868-877.)
© 2005 by Society for Leukocyte Biology

Single gene effects in mouse models of host: pathogen interactions

Anne Fortier, Gundula Min-Oo, John Forbes, Steven Lam-Yuk-Tseung and Philippe Gros1

Department of Biochemistry and Center for the Study of Host Resistance, McGill University, Montreal, Canada

1Correspondence: Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Room 907, Montreal, QC, Canada, H3G-1Y6. E-mail: philippe.gros{at}mcgill.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 THE NATURAL RESISTANCE...
 THE Lgn1 LOCUS
 MALARIA AND THE CHABAUDI...
 CONCLUSION
 REFERENCES
 
Inbred mouse strains have been known for many years to vary in their degree of susceptibility to different types of infectious diseases. The genetic basis of these interstrain differences is sometimes simple but often complex. In a few cases, positional cloning has been used successfully to identify single gene effects. The natural resistance-associated macrophage protein 1 (Nramp1) gene (Slc11a1) codes for a metal transporter active at the phagosomal membrane of macrophages, and Nramp1 mutations cause susceptibility to Mycobacterium, Salmonella, and Leishmania. Furthermore, recent advances in gene transfer technologies in transgenic mice have enabled the functional dissection of gene effects mapping to complex, repeated parts of the genome, such as the Lgn1 locus, causing susceptibility to Legionella pneumophila in macrophages. Finally, complex traits such as the genetically determined susceptibility to malaria can sometimes be broken down into multiple single gene effects. One such example is the case of pyruvate kinase, where a loss-of-function mutation was recently shown by our group to be protective against blood-stage infection with Plasmodium chabaudi. In all three cases reviewed, the characterization of the noted gene effect(s) has shed considerable light on the pathophysiology of the infection, including host response mechanisms.

Key Words: host resistance • genetic


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
THE NATURAL RESISTANCE...
 THE Lgn1 LOCUS
 MALARIA AND THE CHABAUDI...
 CONCLUSION
 REFERENCES
 
The onset, progression, and ultimate outcome of many types of infections are determined by the interaction between defense mechanisms of the host and the capacity of the pathogen to avoid or modify such defenses through expression of so-called virulence or pathogenicity determinants (reviewed in refs. [1 , 2 ]). Genetic studies aimed at identifying mutations causing loss of virulence in vitro or in vivo have proven highly informative to identify microbial proteins and pathways essential for invasion and replication in the host [1 , 2 ]. A similar genetic approach in the host to identify critical defense mechanisms in which malfunction may result in disease has been more challenging as a result of the multitude of physiological systems, cell types, and biochemical pathways involved in host response to pathogens. Nevertheless, a large body of published data suggests that in humans, susceptibility to infectious diseases is influenced by the genetic make-up of the host (reviewed in refs. [3 4 5 ]). In the case of tuberculosis, such evidence includes population studies, unequal geographical distribution of disease, racial differences in susceptibility amongst mixed populations, first contact epidemics, and different disease incidence in monozygotic versus dizygotic twins [3 4 5 ]. One well-documented example of such a genetic effect is malaria, where host genetic determinants appear to affect susceptibility to infection, type of disease developed, and outcome [6 , 7 ]. There is additional evidence of possible co-evolution of the parasite and its human host, including retention of otherwise disease-causing polymorphisms such as sickle cell anemia and ß-thalassemia, which have a protective role against the Plasmodium parasite in areas of endemic disease [6 ]. With the few exceptions of mutations in the interferon-{gamma} and interleukin (IL)-12 pathways, which cause acute mycobacteriosis following bacillus Calmette-Guerin (BCG) vaccination [8 ], CC chemokine receptor 5 deletions conferring protection against AIDS [9 ], and other unique examples [10 ], clear, single gene effects have seldomly been detected in humans. This is a result of, in part, the complex nature of the host:pathogen interaction in general but is more specifically linked to the multigenic and heterogeneous nature of the genetic control, differences in expressivity (health status) of the "susceptibility" and "resistance" determinants, and major pathogen-associated effects such as exposure, dose, and degree of virulence. Conversely, major gene effects can be identified in mouse models of infection [11 12 13 ]. Indeed, excellent models for acute or chronic infection with many pathogens have been developed in the mouse and have been used to study the pathophysiology of the infection and to characterize the critical host response pathways. The ability to control for pathogen effects such as strain, dose, and route of infection is an additional advantage of animal models. Finally, the mouse is an ideal model system for genetic studies, as many inbred, congenic, recombinant inbred, naturally occurring, or experimentally induced mutant stocks are readily available from research labs or commercial suppliers. In addition, the relatively short generation time of the mouse together with the availability of high-density marker maps, bacterial artificial chromosme (BAC) and yeast artificial chromosome contigs, and genome assembly for several mouse strains greatly facilitate genetic analysis including ultimate identification of the gene(s) involved. It is important that the mouse is easily amenable to germ-line modification for the creation of gain- or loss-of-function alleles at individual genes, enabling the formal testing of positional candidates. Such animal studies may identify genes, proteins, and biochemical pathways central to the host defenses [14 15 16 ] and may provide candidate genes for validation in parallel genetic studies in human populations from areas of endemic disease [17 18 19 20 ].

Here, we will review recent work from our laboratory on a few examples of successful cloning of major gene effects in mouse models of infectious diseases. We will also review recent data about the identification of a single gene effect in the context of a complex trait. In all cases, we will highlight the contribution of novel genomics tools that have enabled the identification of these genes.


   THE NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (Nramp1) GENE
TOP
 ABSTRACT
 INTRODUCTION
 THE NATURAL RESISTANCE...
THE Lgn1 LOCUS
 MALARIA AND THE CHABAUDI...
 CONCLUSION
 REFERENCES
 
Over 20 years ago, a locus acting as a major regulator of susceptibility to infection with several intracellular pathogens was identified. This locus, given the appellation Ity, Lsh, and Bcg, was independently detected as modulating susceptibility to infection with Salmonella typhimurium [21 ], Leishmania donovani [22 ], and Mycobacterium bovis (BCG) [23 ], respectively. Ity/Lsh/Bcg is present in two allelic forms—resistance or susceptibility—phenotypically expressed as differential growth rates of the pathogen(s) in the spleen and liver in the early phase of infection. In the case of M. bovis and L. donovani, the susceptible mice ultimately mount an effective immune response and clear the accumulated microbial load, and in the case of virulent S. typhimurium, susceptible animals rapidly succumb to infection. In vitro and in vivo studies have shown that the macrophage is the cell type affected by Bcg/Ity/Lsh [24 25 26 ]. Studies in different inbred mouse strains and in informative F1 backcrosses and F2 progeny derived from them mapped Ity/Lsh/Bcg to mouse chromosome 1 [27 ]. Using a positional cloning approach, we identified the Nramp1 gene as a likely candidate for Bcg/Ity/Lsh [14 ]. The mRNA for Nramp1 is expressed at high levels, almost exclusively in macrophages and neutrophils, and codes for a membrane protein composed of 12 putative transmembrane domains (reviewed in ref. [28 ]). Sequence analyses show that susceptibility to infections in mouse strains is associated with a single glycine-to-aspartate substitution at position 169 in predicted TM4 of the protein, which is associated with a single chromosomal haplotype in inbred strains. Additional studies of gain- or loss-of-function alleles at Nramp1 have established that it is indeed allelic with Ity/Lsh/Bcg [28 ]. Finally, polymorphic variants at or near human NRAMP1 are associated with susceptibility to tuberculosis and leprosy in populations from areas of endemic disease (reviewed in ref. [29 ]).

In phagocytes, the Nramp1 protein is not expressed at the plasma membrane but is rather found in subcellular endomembrane compartments corresponding to lysosomes in macrophages [30 , 31 ] and to gelatinase and vacuolar adenosinetriphosphate (ATPase)-positive tertiary granules in neutrophils [32 ]. Upon phagocytosis, the Nramp1 protein is recruited to the membrane of maturing phagosomes containing inert particles [30 ] or live pathogens [31 , 33 ], where it is proposed to exert its antimicrobial activity (Fig. 1 ). The mechanistic basis of the Nramp1 effect at the phagosomal membrane remained mysterious until the identification and characterization of a second Nramp protein in mammals, Nramp2 [34 ]. Indeed, Nramp-related sequences have been identified in virtually all life forms from bacteria to humans, defining a family of highly conserved membrane transporters [35 , 36 ]. The Nramp2 gene was found to be mutated in the mk mouse [37 ] and the Belgrade rat [38 ], two rodent models of severe iron deficiency and microcytic anemia, characterized by an inability of mutant animals to absorb dietary iron or distribute it for use in peripheral tissues. In parallel, it was demonstrated that the yeast Nramp homologues (Smf1-3) can act to transport metals [39 ]. Finally, direct transport studies established that mammalian Nramp2 (also known as DMT1 and Slc11a2) is a pH-dependent transporter for divalent metals, including Fe2+ and Mn2+, which functions by a proton cotransport mechanism [40 ]. The Nramp2 protein is expressed at the brush border of the duodenum, where it is responsible for dietary iron uptake [41 ]. It is also expressed ubiquitously in peripheral tissues at the plasma membrane and in transferrin receptor-positive recycling endosomes, where it is responsible for transport of transferrin iron across the membrane of acidified endosomes into the cytoplasm [42 ] (Fig. 1B) . By analogy to the known substrate, membrane organization, and direction of transport established for Nramp2, we have proposed a model in which Nramp1 would act as a membrane efflux pump at the membrane of microbe-containing phagosomes, thereby restricting the availability of essential metals such as Mn2+ and Fe2+ to the pathogen [35 , 43 ] (Fig. 1A) . Others have obtained evidence that Nramp1 would act as a metal influx pump at the phagosomal membrane to increase the production of an oxygen radical through the Haber-Weiss reaction [44 , 45 ]. Yet, other studies in Xenopus oocytes have suggested that Nramp1 could act as an antiporter [46 ].



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  		Figure 1. Proposed mechanism of action of Nramp1 (Slc11a1) and Nramp2 (DMT1/Slc11a2). (A) In macrophages, Nramp1 is expressed in lysosomes and is recruited to the membrane of maturing phagosomes containing live bacteria or inert particles. Nramp1 is also expressed in the membrane of gelatinase and H+ vacuolar ATPase-positive tertiary granules of primary neutrophils. We have obtained evidence indicating that Nramp1 functions as a phagosomal metal efflux pump that transports divalent cations Mn2+, Fe2+, and possibly others in a pH-dependent manner and down a proton gradient (acidic inside) generated by the vacuolar H+-ATPase. Nramp1-mediated depletion of divalent cations would inhibit intracellular bacterial replication by removing a metal essential for microbial metabolic activity, by enhancing macrophage bactericidal defenses, or by antagonizing microbial survival functions. (B) Nramp2 is expressed at the plasma membrane of enterocytes, where it mediates Fe2+ acquisition from the duodenum lumen. Nramp2 is also expressed in recycling endosomes and shuttles back and forth to the cell surface. In recycling endosomes, Nramp2 plays a critical role in transport of Fe2+ acquired from the transferrin-transferrin receptor cycle. Upon endosome acidification, transferrin iron is released and becomes available for Nramp2-mediated transport across the endosomal membrane and into the cytosol. The transport of Nramp2 at the plasma membrane and in recycling endosomes is pH-dependent and down a H+ concentration gradient and is abrogated in the mk mouse and Belgrade rat mutants.

 
Recently, we have conducted additional experiments to further characterize the mechanism of metal transport by Nramp1 at the phagosomal membrane, including identifying the substrate specificity and direction of transport [47 48 49 ]. First, we have coupled a metal-sensitive fluorescent dye (Fura-FF6) to solid particles (zymosan), which were then fed to macrophages. Quenching of the probe by extracellular Mn2+ was used to monitor the flux of divalent cations across the phagosomal membrane in permeabilized peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (–/–) mice. Nramp1+/+ phagosomes showed reduced accumulation and increased efflux of Mn2+ (fluorescence quenching) compared with Nramp1–/– phagosomes. The difference in the rate of transport across the phagosomal membrane was eliminated when acidification of the phagosomal lumen was dissipated with bafilomycin, suggesting that divalent metal transport by Nramp1 was H+-dependent, like Nramp2 [47 ]. Another prediction of the functional similarity between Nramp1 and Nramp2 is that should Nramp1 be expressed at the plasma membrane, it would act like Nramp2 and carry out pH-dependent uptake of Fe2+ and Mn2+ from the extracellular medium. Such a Nramp1 variant was created by the insertion of an epitope tag in the predicted extracytoplasmic loop of Nramp1 delineated by predicted TM8 and TM9 [48 ]. Expression of the mutant at the surface of transfected Chinese hamster ovary cells was ascertained by immunofluorescence and by surface biotinylation. Nramp1-HA expression at the cell surface caused pH-dependent uptake of Fe2+ and Mn2+, as measured by fluorescence quenching of calcein and with radioisotopic metals. Transport was pH-dependent and maximal at pH5.5 [48 ]. In these studies, we found that Nramp1 was a more efficient transporter of Mn2+ than Fe2+, suggesting that Mn2+ is the natural substrate for Nramp1 at the phagosomal membrane. These studies strongly suggested that Nramp1 functions as a divalent cations efflux pump at the phagosomal membrane (Fig. 1A) .

Nramp1-mediated divalent metal efflux at the phagosomal membrane has pleiotropic effects on pathogens contained within it. For example, Mycobacteria are known to survive in macrophages by inhibiting phagosome acidification and maturation into phagolysosomes (reviewed in ref. [50 ]). We observed that mycobacterial phagosomes formed in Nramp1-positive macrophages are more acidic than those formed in Nramp1-negative cells and fused more readily to Lamp1-positive lysosomes [51 ]. The reduced acidification seen in Nramp1-negative phagosomes is specific for live mycobacteria and is not seen in Latex bead-containing phagosomes or in phagosomes containing dead mycobacteria. This suggests that Nramp1 may antagonize a biochemical pathway(s) in mycobacteria that is actively responsible for inhibition of phagosome acidification and maturation. Conversely, Salmonella does not inhibit phagosome acidification but rather replicates in a Lamp1-positive, acidified vacuole, which retains certain characteristics of early endosomes. Such Salmonella-containing vacuoles (SCVs) remain largely negative for the mannose-6-phosphate receptor (M6PR). Expression of Nramp1 at the membrane of SCVs was found to stimulate recruitment of M6PR and acquisition of fluid-phase markers introduced via the endocytic pathway [52 ]. These effects could be replicated in Nramp1-negative macrophages by using membrane-permeant iron chelators [49 ]. Together, these results suggest a model in which Nramp1-mediated elimination of divalent cations from the phagosomal space antagonizes the ability of unrelated microbes contained within phagosomes to express their individual survival strategy, including modulation of phagosome maturation.

Therefore, studies of Nramp1 have pointed at a critical role for metals such as iron and manganese in host-pathogen interactions. It is worth noting that intracellular pathogens such as Salmonella possess no less than seven different transport systems for iron (low- or high-affinity, siderophore-dependent or -independent, adenosine 5'-triphosphate, or pH-dependent), including a Nramp-related protein (MntH). Thus, controlling the availability of such metals in the phagosomal space appears to be a critical pleiotropic defense mechanism for the host.


   THE Lgn1 LOCUS
TOP
 ABSTRACT
 INTRODUCTION
 THE NATURAL RESISTANCE...
 THE Lgn1 LOCUS
MALARIA AND THE CHABAUDI...
 CONCLUSION
 REFERENCES
 
Legionella pneumophila causes a severe form of pneumonia in humans, called "Legionnaire’s disease" [53 ]. L. pneumophila is a strict, intracellular pathogen, which replicates in unicellular eukaryotes such as amoebae and tetrahymena, present in its natural aquatic environment, but can also infect tissue macrophages of mammals (reviewed in ref. [54 ]). The bacterium enters macrophages via a unique, so-called "coiled" phagocytosis process and replicates inside a vacuole, which does not mature into fused phagolysosomes but acquires many features of the endoplasmic reticulum, including the presence of ribosomes at the periphery of the phagosomes [54 ]. In human macrophages, replication is associated with induction of apoptosis, a phenomenon not seen with avirulent strains of L. pneumophila (dotA) [55 , 56 ]. Inbred mouse strains are uniformly resistant to L. pneumophila infection with the notable exception of the A/J strain [57 , 58 ]. Using peritoneal macrophages from A/J mice elicited with thioglycollate, Yoshida and his colleagues [59] observed unrestricted replication of L. pneumophila in these cells ex vivo, with a 2- to 3-log increase in bacterial burden over a 72-h period. Using segregation analyses in informative backcrosses and F2 mice derived from A/J and C57BL/6J, they showed that susceptibility to L. pneumophila infection in this experimental setting was controlled by a single locus that they designated Lgn1, which was mapped to the central portion of chromosome 13 by our group [60 ] and others [61 , 62 ]. The gene maps to a complex portion of the mouse genome, which shows conserved synteny with human chromosome 5 in the region containing the gene for spinal muscular atrophy (SMA) [63 , 64 ]. SMA patients show inactivation mutations in the survival motor neuron gene, and some patients show alterations of the nearby neuronal apoptosis inhibitory protein (NAIP) locus [63 , 64 ]. The corresponding mouse chromosomal segment consists of a complex duplication that includes several intact and rearranged copies of the Naip gene. In fact, mouse strains resistant to L. pneumophila such as C57BL/6J and 129X1 harbor different copy numbers of the Naip gene [65 , 66 ]. The repeated and variable nature of the Naip cluster greatly complicates the search for the Lgn1 gene effect by standard methods, including production of a minimal genetic (0.3 cM) and physical (150–300 kb) interval for the gene. Nevertheless, experiments from Growney and Dietrich [65] suggested that the minimal Lgn1 interval must contain only two intact Naip copies, Naip2 and Naip5. Also, we showed that the Naip protein(s) are expressed at high levels in primary macrophages and in macrophage cell lines and that Naip protein(s) are significantly lower in A/J macrophages compared with C57BL/6J macrophages [67 ]. Moreover, it was observed that phagocytosis of L. pneumophila or S. typhimurium or inert particles such as Latex beads result in a further increase in Naip protein expression in these cells. These results suggest that Naip is part of the macrophage response to different "phagocytic" stimuli, making it an attractive candidate for Lgn1 [67 ].

Considering the complexity of the Naip region, we used functional complementation in vivo as a method to identify the gene affected at Lgn1 [68 ]. In these experiments, large genomic clones overlapping the Naip cluster from resistant mice (C57BL/6J, 129X1) and constructed in BACs were introduced in the germ line of transgenic mice otherwise susceptible to infection (A/J) to test for complementing activity [68 ]. In these studies, four overlapping BAC clones of 175–250 kb and covering the Naip locus were selected for microinjection. Unfortunately, the A/J strain is not suitable for production of fertile eggs for microinjection, and thus, fertilized eggs from the FVB strain were used for these studies. However, the FVB strain is resistant to L. pneumophila, requiring further transfer of the transgenic BAC clones from the FVB background to the A/J background by backcrossing. In these experiments, transgenic mice identified by genotyping for BAC vector sequences were backcrossed to A/J mice (Lgn1s) [68 ]. Expression of Naip mRNAs from the transgenic gene copies was monitored at each generation using single nucleotide polymorphisms capable of distinguishing between individual Naip gene copies (i.e., Naip2, Naip5, and others) and the same gene copy from different mouse strains used in the experimental setting (A/J vs. C57BL/6J vs. FVB). Macrophages from such A/J transgenic mice were then tested for their ability to restrict or not intracellular replication of L. pneumophila. Two overlapping BAC clones were found capable of suppressing L. pneumophila replication in this experimental setting, and the effect was transmitted over four generations of A/J backcross. Alignment of the two BAC clones showed that Naip5 was the only intact Naip copy common to both BAC clones [68 ]. Expression studies with macrophages from Legionella-resistant BAC transgenic mice indicated that Naip5 mRNA was overexpressed in these cells, suggesting that Naip5 overexpression could possibly correct haploid insufficiency in A/J mice. Using a similar functional complementation in vivo with Naip BAC clones together with RNA interference inhibition in macrophage cell lines in vitro, Dietrich and his colleagues [69] independently obtained evidence supporting a role of Naip5 in macrophage resistance to L. pneumophila.

What is the biochemical mechanism by which Naip5 can protect macrophages against Legionella infection? Much of the early knowledge about the biochemical function of Naip protein came from their proposed role in inhibiting apoptosis in neuronal cell types [70 ]. Naip proteins (150 kDa) are members of the inhibitor of apoptosis protein family [71 ], structurally defined by so-called baculovirus inhibitor of apoptosis repeat (BIR) domains implicated in protein:protein interactions [72 ]. NAIP also shows a putative nucleotide-binding site [73 ], followed by a carboxy-terminal region seemingly devoid of structural motifs or functional domains. An antiapoptotic effect of NAIP has been described in vitro [71 ] and in vivo [74 ], and inhibition of effector caspases by NAIP BIR domains has been demonstrated [75 ]. In addition, induction of apoptosis appears to be important for pathogenesis of Legionella in human macrophages in vitro [55 , 56 ], and thus, NAIP proteins may act in this pathway. However, Naips have been recently reclassified as members of the nucleotide-binding site (NBS)-leucine-rich repeat (LRR) family of proteins (also known as the "caterpiller" family), which includes Naips, nacht leucine-rich repeat proteins (Nalps), and nucleotide-binding oligomerization domains (NODs; reviewed in refs. [76 , 77 ]; Fig. 2A ). These proteins are structurally defined by the presence of a LRR and a NBS, and each member of this family has a unique signaling module such as the BIR domains for Naip, the CARD domain for NOD1/NOD2, which mediate activation of nuclear factor (NF)-{kappa}B, and the PYD of Nalps [76 , 77 ]. It is important that several of these proteins have been implicated in pattern recognition and intracellular regulation of bacterial-induced inflammation. This has been particularly well-studied in the case of NOD1 and NOD2, which can interact with and can mediate a response to peptidoglycan motifs, diaminopimelate-containing N-acetylglucosamine N-acetylmuramic acid tripeptide motif of Gram-negative bacteria [78 ], and muramyl-dipeptide motif from Gram-negative and -positive bacteria [79 ], respectively. The importance of these proteins in normal host:pathogen interactions in the gut is highlighted by the observation that mutations in human NOD2/CARD15 are associated with chronic inflammatory conditions of the bowel such as Crohn’s disease [80 ] and Blau syndrome [81 ]. In this case, the LRR behaves as a ligand-binding domain, the NBS acts as a dimerization module, and the CARD domain mediates activation of NF-{kappa}B. A LRR is also found in the extracellular domain of another group of well-known pattern recognition proteins, the TLR family. The NBS-LRR family has been highly conserved in evolution with distant members in plants, possibly representing an ancestral mode of recognition of bacterial products. Together, these results suggest that the Naip family of proteins may also be involved in intracellular recognition of bacterial products in macrophages. Naip5 may interact with Legionella-derived products, and this interaction may be disrupted or inefficient in A/J macrophages, a situation corrected in A/J BAC transgenics (Fig. 2B) .



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  		Figure 2. Proposed role of Naip5/Birc1e in macrophage function and resistance to Legionella infection. (A) NAIP5/BIRC1E is structurally related and part of the NBS-LRR or caterpiller family of cytoplasmic proteins. This family is structurally defined by the presence of a NBS, which appears to mediate homodimerization, and a LRR, which is also found in Toll-like receptors (TLRs) and is involved in interaction with bacterial products. Individual members are uniquely defined by a "signaling module" specific to each protein, including caspase recruitment domain (CARD) of NOD1/2, BIR of NAIP5/BIRC1E, the pyrin domain (PYD) of Nalps and CED4, the C. elegans APAF-1 homolog, activation domain (AD) of major histocompatibility complex (MHC) class II transcription activator (CIITA), and the Toll-IL-1 receptor domain (TIR) of plant R protein (for complete review, see ref. [76 ]). Several of these proteins have been shown to interact with products derived from bacteria and to play a critical role in signal transduction in response to these products (see text for details). (B) In normal macrophages from most inbred mouse strains, NAIP5 is proposed to sense the presence of Legionella-derived products; this signaling would be critical in the activation of bactericidal mechanisms of the macrophages to destroy internalized L. pneumophila. In macrophages from the A/J mouse strain, NAIP5 signaling is impaired, and Legionella can inhibit normal phagosome maturation and replicate in a ribosome-studded phagosome that acquires molecular markers of the endoplasmic reticulum.

 
Finally, our studies of the Lgn1 locus show that complex genomic regions can be analyzed effectively by transgenesis with BAC clones for candidate gene regions. We have recently implemented the same BAC transgenesis strategy for the positional cloning of the cytomegalovirus-resistance locus Cmv1 [82 ] and of the neurogenesis mutant Lp [83 ].


   MALARIA AND THE CHABAUDI RESISTANCE LOCUS (Char) FAMILY
TOP
 ABSTRACT
 INTRODUCTION
 THE NATURAL RESISTANCE...
 THE Lgn1 LOCUS
 MALARIA AND THE CHABAUDI...
CONCLUSION
 REFERENCES
 
Although the genetic models of resistance and susceptibility mentioned above are determined by simple traits, which can be identified by positional cloning strategies, the genetic component of susceptibility to many other infections is complex. Tuberculosis and malaria represent two such examples [5 , 6 ]. In such situations, the genetic control is multigenic, and each locus contributes a small fraction of the total interstrain variance. Although quantitative trait locus (QTL) mapping can be used to localize the major gene effects, a fairly large number of animals are required to detect such gene effects, and the corresponding chromosomal regions are often large, containing several hundred candidate genes. One of the strategies to tackle these effects is to use recombinant congenic strains (RCS) of mice, where the genetic complexity can, in some instances, be broken down into simpler gene effects, which can be tackled by more standard methods [84 ].

Malaria is caused by members of the protozoan parasite Plasmodium, and Plasmodium falciparum and Plasmodium vivax are responsible for a large proportion of the human disease [85 ]. Between 300 and 500 million cases of malaria are believed to occur each year, with a reported 1 million fatalities, mostly in young children from impoverished countries. There is no effective vaccine against malaria, and this global health problem has been exacerbated by the development of malarial drug resistance in the Plasmodium parasite and by insecticide resistance in the Anopheles insect vector [85 ]. A large body of published data points to a strong genetic determinant affecting not only innate resistance and susceptibility to infection but also the type of disease developed (cerebral vs. noncomplicated malaria). Polymorphisms, near or at the tumor necrosis factor {alpha} gene, the cell adhesion molecule intercellular adhesion molecule 1, and many others, have been associated with variable susceptibility in association and other studies (reviewed in refs. [6 , 86 ]). It is more striking that polymorphisms in certain major proteins of the erythrocyte (the cell type in which the parasite replicates during the blood stage of the infection) have been shown to have a protective effect against malaria. Indeed, mutations in globin genes (sickle cell anemia, ß-thalassemia) and glucose 6 phosphate dehydrogenase (G6PD) deficiency, although causing debilitating disease in homozygotes, have been shown to exert a malaria-protective effect in heterozygotes, suggesting that positive pressure from the parasite may have caused retention of these mutations in human populations from regions of endemic disease [6 , 86 ]. Our studies have used a murine model of malaria caused by infection with Plasmodium chabaudi AS, which mimics several pathophysiological aspects of the blood-stage infection in humans, including host response and genetic control of parasitemia and ultimate outcome of infection. In this model, the A/J mouse is susceptible, and the C57BL/6J mouse is resistant when using parasitemia at the peak of infection and overall survival as phenotypic measures of susceptibility. Several years ago, we and others mapped a major locus on chromosome 8, designated Char2, which controls peak parasitemia in populations of informative backcross and F2 mice derived between resistant C57BL/6J and susceptible A/J, C3H, and SJL strains [87 , 88 ]. In addition, Foote and his colleagues [87] mapped a robust gene effect on chromosome 9 (Char1), which also affects blood-stage replication of the parasite, and another minor MHC-linked locus (Char3) affects the rate of parasite clearance following the peak of infection [89 ]. Finally, a number of additional, suggestive QTL have been mapped (Table 1 ).


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  		Table 1. Identified Loci Linked to P. chabaudi Susceptibility

 
Recently, we have used RCS of mice derived from A/J and C57BL/6J {[(AXB)XA]XA/[(AXB)XB]XB (AcB/BcA)} to further dissect the complex genetic control of susceptibility to malaria. Systematic inbreeding pairs of AcB and BcA double-backcross mice (N3) produced AcB/BcA strains to generate a set of 15 AcB and 22 BcA strains [94 ]. AcB/BcA strains contain, on average, 12.5% of DNA from one parent fixed as a set of discrete congenic segments on the background (87.5%) of DNA from the other parent. These strains offer a unique opportunity to analyze multigenic traits, as individual resistance/susceptibility loci may independently segregate in individual RCS [94 ]. Phenotyping the AcB/BcA RCS set for resistance to malaria revealed a good overall correlation with the presence of C57BL/6J-derived resistance alleles at Char1 and/or Char2, as measured by peak parasitemia and survival to infection [7 ]. These experiments also identified two discordant strains, AcB55 and AcB61, which showed low parasitemia at the peak of infection with no mortality, despite the presence of A/J susceptibility alleles at Char1 and Char2 [7 ]. Kinetic studies showed that AcB55/61 reached peak parasitemia and cleared the infection even earlier than the malaria-resistant 57BL/6J controls. A partial genome scan to detect a possible association between low peak parasitemia and the presence of 57BL/6J-derived chromosomal fragments was conducted in an informative [AcB55XA]F2. Distribution of peak parasitemia in this F2 cross and in a parallel [AcB61XA]F2 cross suggested that the genetic control of the resistance trait may be complex in both strains. Linkage studies in [AcB55XA]F2 mice identified a significant linkage with a B6-derived segment of chromosome 3 (D3Mit109; LOD=6.57), which was designated Char4 [91 ]. This locus explains ~14% of the variance at peak parasitemia, and 57BL/6J alleles at this locus confer resistance to infection, which is inherited in a recessive manner in males and females. A second, suggestive linkage was mapped to chromosome 10 (D10Mit189; LOD=2.53). Although this QTL was rather weak, it showed an additive effect with Char4 [91 ]. A possible contribution of this Char4 locus to malaria resistance in AcB61 could not be formally evaluated, as this strain contains an A/J-derived chromosome 3, and thus, the [AcB61XA]F2 cross is not informative for this chromosome. No significant linkage on any 57BL/6J-derived, informative chromosomal segment was detected as controlling peak parasitemia in this [AcB61XA]F2 cross [7 ].

Parallel phenotypic studies of the AcB55 and AcB61 strains identified constitutive splenomegaly in these mice, which was further exacerbated following P. chabaudi infection [95 ]. To investigate the molecular basis of splenomegaly and how it could contribute to malaria resistance in these strains, a transcriptional profiling approach was used to identify gene sets that may be differentially regulated in AcB55 and AcB61 strains compared with their A/J parent. This analysis was conducted with cDNA microarrays harboring 15,250 annotated cDNAs to produce a list of genes showing significant differences (>1.5-fold) in transcript abundance in the spleen of AcB55 (n=729) or AcB61 (n=451) compared with A/J, including an overlapping subset of 162 transcripts. Visual inspection of this list of transcripts revealed the presence of two major groups. The first corresponds to erythroid-specific proteins and/or proteins involved in iron metabolism, and the second corresponds to genes involved in cell cycle, DNA replication, and protein synthesis [92 ]. These results suggested increased erythropoiesis in the spleen of AcB55 and AcB61 mice. Parallel analysis of hematological parameters in these mice showed a decreased number of total circulating erythrocytes, an elevated number of Ter119+ cells, and an important increase in the number of circulating reticulocytes (21%), suggesting that AcB55 and AcB61 have severe anemia and constitutive reticulocytosis [95 ]. Segregation analyses in informative [AcB55XA]F2 and [AcB61XA]F2 showed that reticulocytosis was inherited in a recessive manner and segregated as a single locus. In addition, [AcB55XAcB61] mice also showed elevated reticulocyte counts, suggesting a mutation in the same gene or same pathway in both strains [92 ]. Finally, we noted a strong correlation between the high reticulocyte counts and low peak parasitemia in [AcB55XA]F2 and [AcB61XA]F2 mice, suggesting that both traits were related. A formal linkage analysis conducted in fully informative [AcB55XDBA/2]F2 animals showed that reticulocytosis was controlled by a locus on chromosome 3, closely linked to Char4. Inspection of the available transcript maps overlapping the minimal genetic interval identified the erythrocyte and liver-specific pyruvate kinase (pklr) gene as a possible candidate for Char4 [92 ]. PK is absolutely required for glucose metabolism in red cells, and its activity is essential for glucose use and replication of the Plasmodium parasite during the erythrocyte stage of the disease. Sequence analysis identified the presence of a single isoleucine to asparagine (I90N) substitution in the pklr gene of AcB55, which was specific for AcB55 and absent in A/J and C57BL/6J controls and in other commonly used inbred strains. The same sequence alteration was also found in AcB61 genomic DNA. Ile90 is conserved in PKLR from human and other animal species, and the I90N mutation has been previously found in a human case of pyruvate kinase deficiency [96 ]. Haplotype analysis indicated that the I90N mutation arose on the A/J genomic background and became independently fixed during the subsequent inbreeding of strains AcB55 and AcB61. The I90N substitution in Pklr modifies a SfaN I restriction site in genomic DNA, which provides a convenient genomic marker for the mutation. The effect of pklr alleles on susceptibility to malaria was directly assessed in AcB55 and AcB61 F2 crosses to A/J. In both crosses, homozygosity for PklrI90N (–/–) was associated with a significant reduction in the level of parasitemia at the peak of infection, going from 51.6% (for +/+ and +/– controls) to 38.4% parasitized red cells. Homozygosity for PklrI90N significantly reduced mortality of [AcB55XA]F2 mice following P. chabaudi infection, and only 8.5% of –/– mutants succumbed from infection compared with 35% in wild-type heterozygotes and homozygotes. A similar protective effect of PklrI90N homozygosity was also seen in [AcB61XA]F2 mice, and only 15.5% of mutant mice succumbed compared with 65% for controls [92 ]. These results demonstrate that loss-of-function at Pklr (PklrI90N) is associated with reduced blood-stage replication of P. chabaudi and increased survival to murine malaria. Although the molecular basis of the protective effect remains unclear, it may be related to other erythrocyte alterations that have demonstrated malaria-protective effects such as hemoglobin variants and G6PD deficiency. Thus, the mechanism of protection may involve a shortened erythrocyte lifespan, inhospitable environment for intracellular growth, and/or decreased invasion.

Finally, we have obtained evidence indicating that the protective effect of pyruvate kinase deficiency may be further modulated by other host genetic factors. As mentioned, we have mapped a locus (Char9) on chromosome 10 (D10Mit189) that influences the level of peak parasitemia in [AcB55XA]F2 mice, irrespective of heterozygosity or homozygosity for wild-type and mutant alleles at pklr. Conversely, we have identified an AcB strain (AcB62), which despite the presence of protective I90N mutant allele at pklr, shows a susceptibility to malaria with respect to a high degree of blood parasitemia early during infection and by an intermediate rate of mortality, particularly in male AcB62 mice. The positional cloning of these gene effects may reveal other host genes and proteins that play a critical role in pathogenesis or host defenses against the malarial parasite.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 THE NATURAL RESISTANCE...
 THE Lgn1 LOCUS
 MALARIA AND THE CHABAUDI...
 CONCLUSION
REFERENCES
 
A genetic approach in mice to the study of susceptibility to infectious diseases has provided novel insight into the normal mechanisms of host responses to these infections. At the present time, this has been easier to achieve in the case of differential susceptibility traits inherited as monogenic Mendelian traits. The advent of novel approaches for germ-line modification, such as BAC transgenics, has allowed us to extend the positional cloning approach to complex regions that contain multigene families (Lgn1 and Cmv1). Additional novel genetic loci playing an important role in host response to infections and segregating as complex genetic traits have been identified as quantitative trait loci by whole genome scans and in some cases, have been identified. In parallel, several large in vivo N-ethyl-N-nitrosourea mutagenesis experiments have been initiated in different laboratories, and these are likely to reveal yet additional loci affecting host response to pathogens. Therefore, the cloning and characterization of these novel loci during the coming years are likely to shed new light on genes and proteins of the host involved in response to pathogens and ultimately affect the onset, progression, and outcome of infections.


   ACKNOWLEDGEMENTS
 
Research in the P. G. laboratory is supported by research grants from the Canadian Genetic Diseases Network, the Canadian Institutes of Health Research, and the National Institute of Allergy and Infectious Diseases, National Institutes of Health. P. G. is a James McGill Professor of Biochemistry.

Received October 26, 2004; revised December 2, 2004; accepted December 14, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 THE NATURAL RESISTANCE...
 THE Lgn1 LOCUS
 MALARIA AND THE CHABAUDI...
 CONCLUSION
 REFERENCES
 

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