MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage.

Macrophage receptor with collagenous structure (MARCO) has an important role in the phagocytosis of Mycobacterium tuberculosis (M. tuberculosis). We hypothesized that MARCO polymorphisms are associated with phagocytosis, tuberculosis (TB) disease susceptibility and presentation, and infecting lineage. We used a human cellular model to examine how MARCO genotype mediates the immune response; a case-control study to investigate tuberculosis host genetic susceptibility; and a host-pathogen genetic analysis to study host-pathogen interactions. Two MARCO heterozygous (AG) genotypes (single-nucleotide polymorphisms rs2278589 and rs6751745) were associated with impaired phagocytosis of M. tuberculosis trehalose 6,6'-dimycolate-cord factor and ß-glucan-coated beads in macrophages. The heterozygous genotypes of rs2278589 and rs6751745 were also associated with increased risk of pulmonary TB (PTB; rs2278589, P=0.001, odds ratio (OR)=1.6; rs6751745, P=0.009, OR=1.4), and with severe chest X-ray abnormalities (P=0.007, OR=1.6). These two genotypes were also associated with the Beijing lineage (rs2278589, P=0.001, OR=1.7; rs6751745, P=0.01, OR=1.5). Together, these results suggest that MARCO polymorphisms may regulate phagocytosis of M. tuberculosis and susceptibility and severity of PTB. They also suggest MARCO genotype and Beijing strains may interact to increase the risk of PTB.

Small alveolar macrophages are infected preferentially by HIV and exhibit impaired phagocytic function.

HIV-1-infected persons are at higher risk of lower respiratory tract infections than HIV-1-uninfected individuals. This suggests strongly that HIV-infected persons have specific impairment of pulmonary immune responses, but current understanding of how HIV alters pulmonary immunity is incomplete. Alveolar macrophages (AMs), comprising small and large macrophages, are major effectors of innate immunity in the lung. We postulated that HIV-1 impairs pulmonary innate immunity through impairment of AM physiological functions. AMs were obtained by bronchoalveolar lavage from healthy, asymptomatic, antiretroviral therapy-naive HIV-1-infected and HIV-1-uninfected adults. We used novel assays to detect in vivo HIV-infected AMs and to assess AM functions based on the HIV infection status of individual cells. We show that HIV has differential effects on key AM physiological functions, whereby small AMs are infected preferentially by the virus, resulting in selective impairment of phagocytic function. In contrast, HIV has a more generalized effect on AM proteolysis, which does not require direct viral infection. These findings provide new insights into how HIV alters pulmonary innate immunity and the phenotype of AMs that harbors the virus. They underscore the need to clear this HIV reservoir to improve pulmonary immunity and reduce the high incidence of lower respiratory tract infections in HIV-1-infected individuals.

Identification and macrophage-activating activity of glycolipids released from intracellular Mycobacterium bovis BCG.

Intracellular mycobacteria release cell wall glycolipids into the endosomal network of infected macrophages. Here, we characterize the glycolipids of Mycobacterium bovis BCG (BCG) that are released into murine bone marrow-derived macrophages (BMMØ). Intracellularly released mycobacterial lipids were harvested from BMMØ that had been infected with 14C-labelled BCG. Released BCG lipids were resolved by thin-layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides (PIM2), mono- and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono- and dimycolates and the phenolic glycolipid, mycoside B. Culture-derived BCG lipids that co-migrated with the intracellularly released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner similar to release from viable or heat-killed BCG bacilli. To determine whether the released lipids elicited macrophage responses, BCG lipid-coated microspheres were delivered to interferon gamma-primed macrophages (BMMØ or thioglycollate-elicited peritoneal macrophages), and reactive nitrogen intermediates as well as tumour necrosis factor-alpha and monocyte chemoattractant protein-1 production were induced. When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophage-activating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from macrophages that could potentially contribute to the granulomatous response in tuberculous diseases.

Expression of the filarial nematode phosphorylcholine-containing glycoprotein, ES62, is stage specific.

ES62, an immunomodulatory phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae, has previously been shown to be produced by L4 larvae and adult worms only. However, homologous sequences to ES62 have recently been found in L1 and L3 cDNA libraries of certain human filarial nematodes. Therefore, the various stages of A. viteae were re-examined and it was again found that only the post-L3 stages secreted ES62. Synthesis but not secretion by earlier stages was ruled out by examination of the protein content of whole worm extracts and by immunoelectron microscopy. However, examination by PCR of the mRNA for ES62 revealed that it was found in the L1 and L3 larvae. This may explain why homologous sequences to ES62 have been found in Brugia malayi and Onchocerca volvulus larval cDNA libraries. It also suggests that filarial nematodes, in general, may secrete ES62. To obtain evidence for this, we investigated production by Brugia pahangi, a close relation of B. malayi. We found that ES62 was indeed secreted but, as with A. viteae, only by the post-L3 stages, although again the mRNA for ES62 could be detected in the earlier stages. Overall our results suggest that production of ES62 is not species specific, that it is indeed stage specific, and that this may be due to post-transcriptional control of expression.

Mycobacterial persistence: adaptation to a changing environment.

Mycobacterium tuberculosis is a bacterial pathogen that can persist within an infected individual for extended periods of time without causing overt, clinical disease, in a state normally referred to as latent or chronic tuberculosis. Although the replicative state of the bacterium during this period is a matter of some conjecture, recent developments have indicated that the bacterium requires the regulated expression of a set of genes and metabolic pathways to maintain a persistent infection in an immunocompetent host. The characterization of these gene products and their role in bacterial metabolism and physiology is starting to provide insights into the mechanisms that M. tuberculosis has evolved to adopt its highly successful mode of pathogenicity.

Mycobacterium tuberculosis: here today, and here tomorrow.

Mycobacterium tuberculosis is a highly successful pathogen that parasitizes the macrophages of its host. Its success can be attributed directly to its ability to manipulate the phagosome that it resides in and to prevent the normal maturation of this organelle into an acidic, hydrolytic compartment. As the macrophage is key to clearing the infection, the interplay between the pathogen and its host cell reflects a constant battle for control.

Methodology of the 1997 New Zealand National Nutrition Survey.

AIMS: To describe the development and use of the methodologies in the 1997 New Zealand National Nutrition Survey (NNS97). METHODS: NNS97 was a voluntary cross-sectional population survey conducted over a twelve month period on 4,636 non-institutionalized urban and rural New Zealand residents aged fifteen years and older. Survey data were collected in the participants' home and included: a self-administered qualitative food frequency questionnaire, including questions on food preparation habits; a three-pass 24-hour diet recall; interviewer-administered questions on diet supplement use, barriers to dietary change and participant perception of household food security; physical measurements including weight, height, three circumferences, two skinfolds and elbow breadth; blood pressure; and a blood sample to determine cholesterol and iron status. RESULTS: New methodologies developed for NNS97 included a computer based interview system, probability analyses for estimating prevalence of inadequate intake of selected nutrients, determination of iron status by both dietary and biochemical assessment, assessments of dietary supplement use and food security. A full range of quality control procedures at each stage of the data collection were also initiated. CONCLUSION: NNS97 has improved on previous New Zealand national nutrition survey methodologies, particularly with direct data capture and rigorous quality control procedures used in the collection of survey data.

Obesity and body fat distribution in the New Zealand population.

AIMS: To report the prevalence of obesity and body fat distribution in the New Zealand population and to determine if there is a trend to increasing obesity and changes in body fat distribution. METHODS: Body weight, height, two skinfolds (triceps and subscapular), and waist and hip circumferences were measured on 4,420 New Zealanders as part of the 1997 National Nutrition Survey (NNS97). These results are compared with data from the 1977 National Heart Foundation Survey (n=1,800) and the 1989 Life in New Zealand Survey (LINZ89) (n=3,300). RESULTS: 35% of the population (40.4% males, 30.1% females) were classified as overweight and a further 17% as obese (14.7% males, 19.2% females) in NNS97 compared to 32% overweight and 11% obese in LINZ89. Body weight and body mass index have increased in the last two decades. In addition, there has been an increasing trend towards central obesity as estimated by waist to hip ratio and subscapular to triceps ratio. CONCLUSIONS: The increase in body weight, obesity, central obesity, and the proportion of the population likely to exhibit health risk indicators presents an increasing health problem in New Zealand.

Declining levels of total serum cholesterol in adult New Zealanders.

AIM: To measure the average serum concentration of total cholesterol and high density lipoprotein cholesterol in a representative sample of New Zealanders. METHODS: Serum total and high density lipoprotein cholesterol levels were measured in a representative sample of 1,412 men and 1,741 :women aged 15 years or older who participated in the National Nutrition Survey (1997) of New Zealanders. RESULTS: The average serum total cholesterol concentration in men was the same as in women (5.7 mmol/L); however, younger women (44 years and under) tended to have lower levels and older women (55 years and over) higher levels of total cholesterol than men. Women in all age groups had higher average levels of high density lipoprotein cholesterol (1.4 mmol/L) than men (1.2 mmol/L). Ethnic differences were apparent with Maori men having significantly higher average levels of total cholesterol than their New Zealand European counterparts. CONCLUSIONS: Mean serum total cholesterol concentration in women has declined by 0.3 mmol/L from 6.0 mmol/L (p<0.05) since the previous representative survey of New Zealanders (Life in New Zealand Survey, 1989), but by only 0.1 mmol/L in men, despite a similar reduction amongst men and women in the proportion of dietary energy derived from total and saturated fat. It is possible that weight gain in men and women during the last nine years is having a differential effect on serum cholesterol concentrations.

Dietary iron intakes and biochemical iron status of 15-49 year old women in New Zealand: is there a cause for concern?

AIM: To assess dietary iron intakes and biochemical iron status of a nationally representative sample of nonpregnant 15-49 year old women (n=1,751) in New Zealand. METHODS: A cross-sectional national survey was conducted in 1996/97. Women were selected via a multistage stratified cluster sampling procedure with increased sampling of Maori and Pacific women. Dietary iron intakes were estimated using a 24-hour diet recall. Biochemical iron status was assessed on a non-fasting venipuncture blood sample (n=1,047) via haemoglobin, mean cell volume, erythrocyte zinc protoporphyrin, transferrin receptors and serum ferritin. RESULTS: Average daily dietary iron intakes ranged from 9.6 mg/day among Pacific women to 10.5 mg/day among Maori women; 41% of 20-49 year olds and 45% of adolescents were at risk of low dietary iron intakes. The estimated percentage of 15-49 year old women with iron deficiency anaemia ranged from 1.4-5.5%, and for iron deficiency without anaemia from 0.7-12.6% depending on the age group and criteria used. CONCLUSIONS: The overall estimated prevalence of suboptimal biochemical iron status among 15-49 year old women in New Zealand ranged from 7-13%, which compared favourably with premenopausal women living in other western countries. This situation is, however, a public health concern given the potential negative functional consequences associated with even mild iron deficiency.

Attaining optimal bone status: lessons from the 1997 National Nutrition Survey.

AIMS: To examine the adequacy of calcium intake in relation to current recommendations, demographic differences in calcium intake and dietary sources in the New Zealand population. METHODS: 24-hour diet recall and qualitative food frequency data from the 1997 New Zealand National Nutrition Survey (NNS97) were used. RESULTS: No age-gender subgroups had median intakes meeting the latest (1998) US recommendations. Women's median intakes failed to meet even the considerably lower 1990 Australian recommendations. 20% of New Zealanders and one in four women had intakes below the UK Estimated Average Requirements for calcium. Intakes below the UK Lower Reference Nutrient Intake (the level at which the risk of deficiency is virtually 100%) were common (15-20%) among women aged 15-18 years, those living in the most deprived areas or Maori. Milk and milk products were the major sources of the nation's calcium intake. CONCLUSION: Although other factors such as genetics, hormonal status, vitamin D status and exercise influence skeletal health, adequate calcium intakes are important in minimizing bone loss. A reduction in the proportion of New Zealanders with inadequate calcium intakes will most readily be achieved if more people meet the milk products Dietary Guideline (minimum of two servings daily). Health professionals can play an important role in raising perceptions of the benefits of adequate calcium intakes, promoting the milk products Dietary Guideline, and emphasising that lower fat diets can include adequate calcium through use of reduced fat milk products.

Food security: is New Zealand a land of plenty?

AIM: To explore the concept of food security (when there is enough, appropriate and acceptable food available) in the adult New Zealand population using the National Nutrition Survey (NNS97). METHODS: The stepwise development of indicators of food security included a literature search and focus groups with low income women and men. Key issues surrounding the procurement and provision of food were determined and eight indicator statements prepared for inclusion in NNS97, to be addressed by each participant on behalf of their household. RESULTS: Prevalence was significantly higher (p<0.05) for females compared to males for the majority of indicator statements among New Zealand European & Others and Maori. New Zealand European & Others reported the most food security; Pacific people reported the least and Maori fell between the two. There was a significant increasing linear trend of food security with age (p<0.001), after adjusting for gender. "Food runs out in my/our household due to lack of money" was cited more often by female compared to male New Zealand European & Others in NZDep96 quartiles III and IV. CONCLUSION: The issue of 'not having enough food' may be more prevalent in New Zealand than US or Australia. Among New Zealand European & Others the higher prevalence of insufficient food due to lack of money among females from NZDep96 quartiles III and IV suggests that males may be protected from this by their female partners. Food security needs to be improved among young adults, women, Maori and Pacific people in order to prevent longer term nutritional health consequences.

Mycobacterial surface moieties are released from infected macrophages by a constitutive exocytic event.

Bacterial cell wall constituents are released from mycobacterial phagosomes and actively traffic within infected macrophages. Colocalization of fluorescently tagged bacterial moieties with endocytic tracers revealed the dynamic movement of released mycobacterial constituents into the endocytic network with accumulation in tubular lysosomal-like compartments. The released bacterial constituents not only penetrated the infected host cell but were also present in an extracellular microvesicular fraction. To identify the intracellular source of these exocytic compartments, released vesicular material was isolated from culture supernatants by differential ultracentrifugation and characterized by Western blot and electron microscopy analyses. The presence of lysosomal membrane proteins and lysosomal proteases suggested that labeled mycobacterial cell wall constituents access a constitutive lysosomal exocytic pathway. An abundance of multilamellar extracellular compartments morphologically reminiscent of MHC class II-enriched compartments (MIIC) implicated a MHC class II transport pathway in the extracellular release of bacterial constituents. Increases in intracellular free calcium have previously been shown to trigger lysosomal exocytosis by inducing fusion of lysosomes with the plasma membrane. To test if an increase in calcium would stimulate exocytosis with release of mycobacterial constituents, infected macrophages were exposed to the calcium ionophore A23187. The ionophore triggered the release of a microvesicular fraction containing labeled bacterial moieties, implicating calcium-regulated lysosomal exocytosis as a trafficking pathway by which mycobacterial products are released from infected macrophages.

Analysis of mycobacterium-infected macrophages by immunoelectron microscopy and cell fractionation.

The ability of pathogenic Mycobacterium to establish and maintain an infection in a host is dependent on their capacity to survive within phagocytes (1-3). Studies conducted on macrophage infections in culture have provided considerable insight into the mechanisms developed by these bacteria to ensure their survival. However, macrophages in culture are considerably more permissive than the phagocyte in its correct tissue environment and one has to be aware that the capacity of the macrophage to function as both an antigen-presenting cell (inducing or sustaining a cellular immune response) and an immune effector cell (mediating an antimicrobial response following activation with cytokines) places certain provisos on interpretation of in vitro infection experiments (4,5). Despite this obvious caveat, our appreciation of the complex interplay between Mycobacterium spp. of varying degrees of virulence [M. tuberculosis, M. bovis (BCG) and M. avium] and their host macrophage has benefited considerably from the recent application of modern cell biological techniques to studies of infected cells in culture.

Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages.

Considerable effort has focused on the identification of proteins secreted from Mycobacterium spp. that contribute to the development of protective immunity. Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages. In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed. Mycobacterium bovis BCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe. The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages. Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes. To identify these proteins, subcellular fractions free of bacteria were probed with mycobacterium-specific antibodies. The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome.

Interaction of Mycobacterium avium-containing phagosomes with the antigen presentation pathway.

Pathogenic mycobacteria infect macrophages where they replicate in phagosomes that minimize contact with late endosomal/lysosomal compartments. Loading of Ags to MHC class II molecules occurs in specialized compartments with late endosomal characteristics. This points to a sequestration of mycobacteria-containing phagosomes from the sites where Ags meet MHC class II molecules. Indeed, in resting macrophages MHC class II levels decreased strongly in phagosomes containing M. avium during a 4-day infection. Phagosomal MHC class II of early (4 h) infections was partly surface-derived and associated with peptide. Activation of host macrophages led to the appearance of H2-M, a chaperon of Ag loading, and to a strong increase in MHC class II molecules in phagosomes of acute (1 day) infections. Comparison with the kinetics of MHC class II acquisition by IgG-coated bead-containing phagosomes suggests that the arrest in phagosome maturation by mycobacteria limits the intersection of mycobacteria-containing phagosomes with the intracellular trafficking pathways of Ag-presenting molecules.

Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase.

Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Despite an urgent need for new therapies targeting persistent bacteria, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of fatty acids. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. A link between the requirement for ICL and the immune status of the host was established by the restored virulence of delta icl bacteria in interferon-gamma knockout mice. This link was apparent at the level of the infected macrophage: Activation of infected macrophages increased expression of ICL, and the delta icl mutant was markedly attenuated for survival in activated but not resting macrophages. These data suggest that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection, an observation with important implications for the treatment of chronic tuberculosis.

Structure of isocitrate lyase, a persistence factor of Mycobacterium tuberculosis.

Isocitrate lyase (ICL) plays a pivotal role in the persistence of Mycobacterium tuberculosis in mice by sustaining intracellular infection in inflammatory macrophages. The enzyme allows net carbon gain by diverting acetyl-CoA from beta-oxidation of fatty acids into the glyoxylate shunt pathway. Given its potential as a drug target against persistent infections, we solved its structure without ligand and in complex with two inhibitors. Covalent modification of an active site residue, Cys 191, by the inhibitor 3-bromopyruvate traps the enzyme in a catalytic conformation with the active site completely inaccessible to solvent. The structure of a C191S mutant of the enzyme with the inhibitor 3-nitropropionate provides further insight into the reaction mechanism.

Sequence requirements for trafficking of the CRAM transmembrane protein to the flagellar pocket of African trypanosomes.

CRAM is a cysteine-rich acidic transmembrane protein, highly expressed in the procyclic form of Trypanosoma brucei. Cell surface expression of CRAM is restricted to the flagellar pocket of trypanosomes, the only place where receptor mediated endocytosis takes place in the parasite. CRAM can function as a receptor and was hypothesized to be a lipoprotein receptor of trypanosomes. We study mechanisms involved in the presentation and routing of CRAM to the flagellar pocket of insect- and bloodstream-form trypanosomes. By deletional mutagenesis, we found that deleting up to four amino acids from the C terminus of CRAM did not affect the localization of CRAM at the flagellar pocket. Shortening the CRAM protein by 8 and 19 amino acids from the C terminus resulted in the distribution of the CRAM protein in the endoplasmic reticulum (ER) (the CRAM protein is no longer uniquely sequestered at the flagellar pocket). This result indicates that the truncation of the CRAM C terminus affected the transport efficiency of CRAM from the ER to the flagellar pocket. However, when CRAM was truncated between 29 and 40 amino acids from the C terminus, CRAM was not only distributed in the ER but also located to the flagellar pocket and spread to the cell surface and the flagellum. Replacing the CRAM transmembrane domain with the invariant surface glycoprotein 65-derived transmembrane region did not affect the flagellar pocket location of CRAM. These results indicate that the CRAM cytoplasmic extension may exhibit two functional domains: one domain near the C terminus is important for efficient export of CRAM from the ER, while the second domain is of importance for confining CRAM to the flagellar pocket membrane.