Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO₂⁻ binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO₂⁻ concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO₂−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.     

Truncated structural variants of lipoarabinomannan in Mycobacterium leprae and an ethambutol-resistant strain of Mycobacterium tuberculosis

Current knowledge on the structure of lipoarabinomannan (LAM) has resulted primarily from detailed studies on a few selected laboratory strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmatis. Our previous work was the first to report on the salient structural features of M. tuberculosis clinical isolates and demonstrated significant structural variations. A prime effort is to correlate a particular structural characteristic with observed differences in eliciting an immunobiological response, especially in the context of CD1-restricted presentation of LAM to T cells. T cell clones derived from the cutaneous lesions of leprosy patients have been shown to recognize specifically LAM from Mycobacterium leprae and not from M. tuberculosis Erdman or H37Rv. Herein we provide further fine structural data on LAM from M. leprae (LepLAM) and a tuberculosis clinical isolate, CSU20 (CSU20LAM), which was unexpectedly recognized by the supposedly LepLAM-specific CD1-restricted T cell clones. In comparison with the de facto laboratory LAM standard from M. tuberculosis H37Rv (RvLAM), LepLAM derived from in vivo grown M. leprae is apparently simpler in its arabinan architecture with a high degree of exposed, non-mannose-capped termini. On the other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly heterogeneous population of LAM ranging from rather small and non-mannose-capped to full-length and fully capped variants. LepLAM and CSU20LAM contain a higher level of succinylation than RvLAM, which, in the context of truncated or less elaborated arabinan, may contribute to selective recognition by T cells. LAM from all species could be resolved into discrete forms by isoelectric focusing based apparently on their arabinan heterogeneity. In the light of our current and more recent findings, we reason that all immunobiological data should be cautiously interpreted and that the actual LAM variants that may be present in vivo during infection and pathogenesis need to be taken into consideration.     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

The Evolution of Campylobacter jejuni and Campylobacter coli

The global significance of Campylobacter jejuni and Campylobacter coli as gastrointestinal human pathogens has motivated numerous studies to characterize their population biology and evolution. These bacteria are a common component of the intestinal microbiota of numerous bird and mammal species and cause disease in humans, typically via consumption of contaminated meat products, especially poultry meat. Sequence-based molecular typing methods, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS), have been instructive for understanding the epidemiology and evolution of these bacteria and how phenotypic variation relates to the high degree of genetic structuring in C. coli and C. jejuni populations. Here, we describe aspects of the relatively short history of coevolution between humans and pathogenic Campylobacter, by reviewing research investigating how mutation and lateral or horizontal gene transfer (LGT or HGT, respectively) interact to create the observed population structure. These genetic changes occur in a complex fitness landscape with divergent ecologies, including multiple host species, which can lead to rapid adaptation, for example, through frame-shift mutations that alter gene expression or the acquisition of novel genetic elements by HGT. Recombination is a particularly strong evolutionary force in Campylobacter, leading to the emergence of new lineages and even large-scale genome-wide interspecies introgression between C. jejuni and C. coli. The increasing availability of large genome datasets is enhancing understanding of Campylobacter evolution through the application of methods, such as genome-wide association studies, but MLST-derived clonal complex designations remain a useful method for describing population structure.Campylobacter jejuni and Campylobacter coli remain among the most common causes of human bacterial gastroenteritis worldwide (Friedman et al. 2000). In high-income countries, Campylobacteriosis is much more common than gastroenteritis caused by Escherichia coli, Listeria, and Salmonella, and accounts for an estimated 2.5 million annual cases of gastrointestinal disease in the United States alone (Kessel et al. 2001). Infection with these bacteria is also a major cause of morbidity and mortality in low- and middle-income countries, although it is almost certainly underreported in these settings, especially as culture confirmation remains challenging. Poor understanding of the transmission of these food-borne pathogens to humans in all income settings has contributed to the failure of public health systems to adequately address this problem. As a consequence, over the past 20 years, much investment has been directed at understanding how these bacteria are transmitted from reservoir hosts to humans through the food chain.Although the disease was first recognized by Theodor Escherich in 1886, who described the symptoms of intestinal Campylobacter infections in children as “cholera infantum” (Samie et al. 2007) or “summer complaint” (Condran and Murphy 2008), difficulties in the culture and characterization of these organisms precluded their recognition as major causes of disease until the 1970s. Campylobacteriosis is usually nonfatal and self-limiting; however, the symptoms of diarrhea, fever, abdominal pain, and nausea can be severe (Allos 2001), and sequelae, including Guillain–Barre syndrome and reactive arthritis, can have serious long-term consequences. Subsequently, recognition of the very high disease burden of human Campylobacter infection stimulated research on these bacteria and their relatives. Since the 1970s, C. coli and C. jejuni have been isolated from a wide range of wild and domesticated bird and mammal species, in which, typically, they are thought to cause few if any disease symptoms. Humans are usually infected by the consumption of contaminated food (especially poultry meat), water, milk, or contact with animals or animal feces (Niemann et al. 2003).Most of what is known about these species comes from isolates obtained from humans with disease, the food chain, and the agricultural environment. It is, however, important to note that such isolates are by no means representative of natural Campylobacter populations, and it is becoming increasingly apparent that much of the diversity present among the Campylobacters is in strains that colonize wild animals. Increasing numbers of novel genotypes are being found as Campylobacter populations are analyzed in different animal species, especially wild birds (Carter et al. 2009; French et al. 2009); these populations undoubtedly contain many as-yet-undescribed lineages. Most human disease isolates from cases of gastroenteritis in countries, such as the United Kingdom and the United States, are C. jejuni, which typically accounts for 90% of cases in these settings, with the remaining ∼10% of cases mostly caused by C. coli. The majority of the genotypes isolated from human disease have also been isolated as commensal gastrointestinal inhabitants of domesticated and, especially, food animals. Furthermore, clinical isolates are a nonrandom subset of these strains. Asymptomatic carriage of C. jejuni and C. coli is thought to be rare in humans, especially among people in industrialized countries, suggesting that humans are not a primary host for these organisms in these settings and that people are sporadically, and frequently pathologically, infected via the food chain from animal reservoir hosts.An understanding of the relatively short history of coevolution between humans and pathogenic Campylobacters can be obtained by examining their population structure and ecology. This approach has formed the basis of many recent investigations of the cryptic epidemiology of these organisms (Lang et al. 2010; Müllner et al. 2010; Thakur et al. 2010; Hastings et al. 2011; Jorgensen et al. 2011; Kittl et al. 2011; Magnússon et al. 2011; Sheppard et al. 2011a,b; Sproston et al. 2011; Read et al. 2013) and will be the focus of this review. Such studies have included molecular epidemiological and evolutionary analyses and, in the past 15 years or so, the application of high-throughput DNA sequencing technologies of increasing capacity has enhanced the integration of these two areas of investigation to their mutual benefit.     

Role of PheE15 Gate in Ligand Entry and Nitric Oxide Detoxification Function of Mycobacterium tuberculosis Truncated Hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O₂ and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O₂/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.     

结核分枝杆菌耐氟喹诺酮类药物的分子机制研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis)通过空气传播引起人类感染的慢性传染病,耐药结核分枝杆菌的流行是目前结核病防治的世界难题。氟喹诺酮类药物是人工合成药物,应用于耐药结核的临床治疗中,在治疗中起着核心的作用。但近年来,氟喹诺酮类药物的抗性菌株不断出现,愈发增加了结核病治疗的困难与治疗失败风险。在临床中氟喹诺酮药物的靶点比较清楚,是结核分枝杆菌的DNA旋转酶。目前发现结核分枝杆菌耐氟喹诺酮类药物的机制主要包括药物靶点DNA旋转酶的关键氨基酸改变、药物外排泵系统、细菌细胞壁厚度的增加以及喹诺酮抗性蛋白MfpA介导的DNA旋转酶活性调控。其中在氟喹诺酮靶标DNA旋转酶功能活性改变的耐药机制方面,编码DNA旋转酶基因突变一直是研究的热点,但近年来发现DNA旋转酶的调控蛋白MfpA以及DNA旋转酶的修饰在细菌耐药性中起着重要的作用,相关机制还亟待发现。本文综述了当前结核分枝杆菌耐氟喹诺酮类药物的作用机制,旨在为研发精准诊断技术和药物发掘提供科学理论基础和参考。     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals.

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

Genome maps of Campylobacter jejuni and Campylobacter coli.

Little information concerning the genome of either Campylobacter jejuni or Campylobacter coli is available. Therefore, we constructed genomic maps of C. jejuni UA580 and C. coli UA417 by using pulsed-field gel electrophoresis. The genome sizes of C. jejuni and C. coli strains are approximately 1.7 Mb, as determined by SalI and SmaI digestion (N. Chang and D. E. Taylor, J. Bacteriol. 172:5211-5217, 1990). The genomes of both species are represented by single circular DNA molecules, and maps were constructed by partial restriction digestion and hybridization of DNA fragments extracted from low-melting-point agarose gels. Homologous DNA probes, encoding the flaAB and 16S rRNA genes, as well as heterologous DNA probes from Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, were used to identify the locations of particular genes. C. jejuni and C. coli contain three copies of the 16S and 23S rRNA genes. However, they are not located together within an operon but show a distinct split in at least two of their three copies. The positions of various housekeeping genes in both C. jejuni UA580 and C. coli UA417 have been determined, and there appears to be some conservation of gene arrangement between the two species.     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

Colonization of Cattle Intestines by Campylobacter jejuni and Campylobacter lanienae

The location and abundance of Campylobacter jejuni and Campylobacter lanienae in the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shed C. jejuni and C. lanienae (steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed. Campylobacter DNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. Although C. jejuni and C. lanienae DNA were detected in both animals, only steer A contained substantial quantities of C. jejuni DNA. In both digesta and tissues of steer A, C. jejuni was present in the duodenum and jejunum. Considerable quantities of C. jejuni DNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities of C. lanienae DNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associated C. lanienae DNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance of C. jejuni and C. lanienae DNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA of C. jejuni and C. lanienae were detected in the digesta of 57% and 95% of the animals, respectively. C. jejuni associated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance of C. jejuni DNA in the ileum and rectum. C. lanienae was most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore, C. jejuni colonized the proximal small intestine of asymptomatic beef cattle, whereas C. lanienae primarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.     

Colonization of cattle intestines by Campylobacter jejuni and Campylobacter lanienae

The location and abundance of Campylobacter jejuni and Campylobacter lanienae in the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shed C. jejuni and C. lanienae (steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed. Campylobacter DNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. Although C. jejuni and C. lanienae DNA were detected in both animals, only steer A contained substantial quantities of C. jejuni DNA. In both digesta and tissues of steer A, C. jejuni was present in the duodenum and jejunum. Considerable quantities of C. jejuni DNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities of C. lanienae DNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associated C. lanienae DNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance of C. jejuni and C. lanienae DNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA of C. jejuni and C. lanienae were detected in the digesta of 57% and 95% of the animals, respectively. C. jejuni associated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance of C. jejuni DNA in the ileum and rectum. C. lanienae was most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore, C. jejuni colonized the proximal small intestine of asymptomatic beef cattle, whereas C. lanienae primarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle.

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

Prevalence of Campylobacter jejuni,Campylobacter lari,and Campylobacter coli in different ecological guilds and taxa of migrating birds

A total of 1,794 migrating birds trapped at a coastal site in southern Sweden were sampled for detection of Campylobacter spp. All isolates phenotypically identified as Campylobacter jejuni and a subset of those identified as non-C. jejuni were identified to the species level by PCR-based techniques. C. jejuni was found in 5.0% of the birds, Campylobacter lari was found in 5.6%, and Campylobacter coli was found in 0.9%. An additional 10.7% of the tested birds were infected with hippurate hydrolysis-negative Campylobacter spp. that were not identified to the species level. The prevalence of Campylobacter spp. differed significantly between ecological guilds of birds. Shoreline-foraging birds feeding on invertebrates and opportunistic feeders were most commonly infected (76.8 and 50.0%, respectively). High prevalence was also shown in other ground-foraging guilds, i.e., ground-foraging invertebrate feeders (11.0%), ground-foraging insectivores (20.3%), and plant-eating species (18.8%). Almost no Campylobacter spp. were found in ground-foraging granivores (2.3%), arboreal insectivores (0.6%), aerial insectivores (0%), or reed- and herbaceous plant-foraging insectivores (3.5%). During the autumn migration, a high proportion of samples from juveniles were positive (7.1% in passerines, 55.0% in shorebirds), indicating transmission on the breeding grounds or during the early part of migration. Prevalence of Campylobacter spp. was associated with increasing body mass among passerine bird species. Furthermore, prevalence was higher in short-distance migrants wintering in Europe than in long-distance migrants wintering in Africa, the Middle East, or Asia. Among ground-foraging birds of the Muscicapidae, those of the subfamily Turdinae (i.e., Turdus spp.) showed a high prevalence of Campylobacter spp., while the organism was not isolated in any member of the subfamily Muscicapinae (i.e., Erithacus and Luscinia). The prevalence of Campylobacter infection in wild birds thus seems to be linked to various ecological and phylogenetic factors, with great variations in carriership between different taxa and guilds.     

Truncated Hemoglobin,HbN, Is Post-translationally Modified in Mycobacterium tuberculosis and Modulates Host-Pathogen Interactions during Intracellular Infection

Mycobacterium tuberculosis (Mtb) is a phenomenally successful human pathogen having evolved mechanisms that allow it to survive within the hazardous environment of macrophages and establish long term, persistent infection in the host against the control of cell-mediated immunity. One such mechanism is mediated by the truncated hemoglobin, HbN, of Mtb that displays a potent O₂-dependent nitric oxide dioxygenase activity and protects its host from the toxicity of macrophage-generated nitric oxide (NO). Here we demonstrate for the first time that HbN is post-translationally modified by glycosylation in Mtb and remains localized on the cell membrane and the cell wall. The glycan linkage in the HbN was identified as mannose. The elevated expression of HbN in Mtb and M. smegmatis facilitated their entry within the macrophages as compared with isogenic control cells, and mutation in the glycan linkage of HbN disrupted this effect. Additionally, HbN-expressing cells exhibited higher survival within the THP-1 and mouse peritoneal macrophages, simultaneously increasing the intracellular level of proinflammatory cytokines IL-6 and TNF-α and suppressing the expression of co-stimulatory surface markers CD80 and CD86. These results, thus, suggest the involvement of HbN in modulating the host-pathogen interactions and immune system of the host apart from protecting the bacilli from nitrosative stress inside the activated macrophages, consequently driving cells toward increased infectivity and intracellular survival.     

Explorative Multifactor Approach for Investigating Global Survival Mechanisms of Campylobacter jejuni under Environmental Conditions

Explorative approaches such as DNA microarray experiments are becoming increasingly important in microbial research. Despite these major technical advancements, approaches to study multifactor experiments are still lacking. We have addressed this problem by using rotation testing and a novel multivariate analysis of variance (MANOVA) approach (50-50 MANOVA) to investigate interacting experimental factors in a complex experimental design. Furthermore, a new rotation testing based method was introduced to calculate false-discovery rates for each response. This novel analytical concept was used to investigate global survival mechanisms in the environment of the major food-borne pathogen C. jejuni. We simulated nongrowth environmental conditions by investigating combinations of the factors temperature (5 and 25°C) and oxygen tension (anaerobic, microaerobic, and aerobic). Data were generated with DNA microarrays for information about gene expression patterns and Fourier transform infrared (FT-IR) spectroscopy to study global macromolecular changes in the cell. Microarray analyses showed that most genes were either unchanged or down regulated compared to the reference (day 0) for the conditions tested and that the 25°C anaerobic condition gave the most distinct expression pattern with the fewest genes expressed. The few up-regulated genes were generally stress related and/or related to the cell envelope. We found, using FT-IR spectroscopy, that the amount of polysaccharides and oligosaccharides increased under the nongrowth survival conditions. Potential mechanisms for survival could be to down regulate most functions to save energy and to produce polysaccharides and oligosaccharides for protection against harsh environments. Basic knowledge about the survival mechanisms is of fundamental importance in preventing transmission of this bacterium through the food chain.     

Hemoglobins of baboons, Papio cynocephalus, P. gelada and P. hamadryas

Major hemoglobins of adult Papio cynocephalus, P. gelada, and P. hamadryas and of newborn P. cynocephalus were purified; globins were prepared; and α and β or α and γ chains were separated. The amino acid compositions and aminoterminal groups were determined. These were compared with analogous data for human hemoglobins, other baboon hemoglobins and macaque hemoglobins.     

The bactericidal activity of tea against Campylobacter jejuni and Campylobacter coli

Extracts of black and green tea inhibited the growth of clinical isolates of Campylobacter jejuni and C. coli. Tea extracts killed C. jejuni and C. coli within 4 h. Heat treatment of extracts did not affect inhibitory or bactericidal activity.