IgA-deficient humans exhibit gut microbiota dysbiosis despite secretion of compensatory IgM.

Immunoglobulin A is the dominant antibody isotype found in mucosal secretions and enforces host-microbiota symbiosis in mice, yet selective IgA-deficiency (sIgAd) in humans is often described as asymptomatic. Here, we determined the effects of IgA deficiency on human gut microbiota composition and evaluated the possibility that mucosal secretion of IgM can compensate for a lack of secretory IgA. We used 16S rRNA gene sequencing and bacterial cell sorting to evaluate gut microbiota composition and taxa-specific antibody coating of the gut microbiota in 15 sIgAd subjects and matched controls. Despite the secretion of compensatory IgM into the gut lumen, sIgAd subjects displayed an altered gut microbiota composition as compared to healthy controls. These alterations were characterized by a trend towards decreased overall microbial diversity as well as significant shifts in the relative abundances of specific microbial taxa. While secretory IgA in healthy controls targeted a defined subset of the microbiota via high-level coating, compensatory IgM in sIgAd subjects showed less specificity than IgA and bound a broader subset of the microbiota. We conclude that IgA plays a critical and non-redundant role in controlling gut microbiota composition in humans and that secretory IgA has evolved to maintain a diverse and stable gut microbial community.

Human gut Bacteroides capture vitamin B12 via cell surface-exposed lipoproteins.

Human gut Bacteroides use surface-exposed lipoproteins to bind and metabolize complex polysaccharides. Although vitamins and other nutrients are also essential for commensal fitness, much less is known about how commensal bacteria compete with each other or the host for these critical resources. Unlike in Escherichia coli, transport loci for vitamin B12 (cobalamin) and other corrinoids in human gut Bacteroides are replete with conserved genes encoding proteins whose functions are unknown. Here we report that one of these proteins, BtuG, is a surface-exposed lipoprotein that is essential for efficient B12 transport in B. thetaiotaomicron. BtuG binds B12 with femtomolar affinity and can remove B12 from intrinsic factor, a critical B12 transport protein in humans. Our studies suggest that Bacteroides use surface-exposed lipoproteins not only for capturing polysaccharides, but also to acquire key vitamins in the gut.

Gut Microbiota: IgA Protects the Pioneers.

The secretory antibody immunoglobulin A counteracts pathogenic infections at mucosal surfaces. Recent work now reveals that IgA responses can also stabilize intestinal colonization by symbiotic microorganisms and confer resistance to future invasion by exogenous competitors.

The Stringent Response Determines the Ability of a Commensal Bacterium to Survive Starvation and to Persist in the Gut.

In the mammalian gut, bacteria compete for resources to maintain their populations, but the factors determining their success are poorly understood. We report that the human gut bacterium Bacteroides thetaiotaomicron relies on the stringent response, an intracellular signaling pathway that allocates resources away from growth, to survive carbon starvation and persist in the gut. Genome-scale transcriptomics, 13C-labeling, and metabolomics analyses reveal that B. thetaiotaomicron uses the alarmone (p)ppGpp to repress multiple biosynthetic pathways and upregulate tricarboxylic acid (TCA) cycle genes in these conditions. During carbon starvation, (p)ppGpp triggers accumulation of the metabolite alpha-ketoglutarate, which itself acts as a metabolic regulator; alpha-ketoglutarate supplementation restores viability to a (p)ppGpp-deficient strain. These studies uncover how commensal bacteria adapt to the gut by modulating central metabolism and reveal that halting rather than accelerating growth can be a determining factor for membership in the gut microbiome.

Dot/Icm-Translocated Proteins Important for Biogenesis of the Coxiella burnetii-Containing Vacuole Identified by Screening of an Effector Mutant Sublibrary.

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. A determinant necessary for C. burnetii virulence is the Dot/Icm type IVB secretion system (T4SS). The Dot/Icm system delivers more than 100 proteins, called type IV effectors (T4Es), across the vacuolar membrane into the host cell cytosol. Several T4Es have been shown to be important for vacuolar biogenesis. Here, transposon (Tn) insertion sequencing technology (INSeq) was used to identify C. burnetii Nine Mile phase II mutants in an arrayed library, which facilitated the identification and clonal isolation of mutants deficient in 70 different T4E proteins. These effector mutants were screened in HeLa cells for deficiencies in Coxiella-containing vacuole (CCV) biogenesis. This screen identified and validated seven new T4Es that were important for vacuole biogenesis. Loss-of-function mutations in cbu0414 (coxH1), cbu0513, cbu0978 (cem3), cbu1387 (cem6), cbu1524 (caeA), cbu1752, or cbu2028 resulted in a small-vacuole phenotype. These seven mutant strains produced small CCVs in all cells tested, which included macrophage-like cells. The cbu2028::Tn mutant, though unable to develop large CCVs, had intracellular replication rates similar to the rate of the parental strain of C. burnetii, whereas the other six effector mutants defective in CCV biogenesis displayed significant reductions in intracellular replication. Vacuoles created by the cbu0513::Tn mutant did not accumulate lipidated microtubule-associated protein 1A/1B light chain 3 (LC3-II), suggesting a failure in fusion of the CCV with autophagosomes. These seven T4E proteins add to the growing repertoire of C. burnetii factors that contribute to CCV biogenesis.

Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries.

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.

Human symbionts inject and neutralize antibacterial toxins to persist in the gut.

The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes--one of two major phyla in the gut--also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.

Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics.

Single-molecule fluorescence imaging is at the forefront of tools applied to study biomolecular dynamics both in vitro and in vivo. The ability of the single-molecule fluorescence microscope to conduct simultaneous multi-color excitation and detection is a key experimental feature that is under continuous development. In this paper, we describe in detail the design and the construction of a sophisticated and versatile multi-color excitation and emission fluorescence instrument for studying biomolecular dynamics at the single-molecule level. The setup is novel, economical and compact, where two inverted microscopes share a laser combiner module with six individual laser sources that extend from 400 to 640 nm. Nonetheless, each microscope can independently and in a flexible manner select the combinations, sequences, and intensities of the excitation wavelengths. This high flexibility is achieved by the replacement of conventional mechanical shutters with acousto-optic tunable filter (AOTF). The use of AOTF provides major advancement by controlling the intensities, duration, and selection of up to eight different wavelengths with microsecond alternation time in a transparent and easy manner for the end user. To our knowledge this is the first time AOTF is applied to wide-field total internal reflection fluorescence (TIRF) microscopy even though it has been commonly used in multi-wavelength confocal microscopy. The laser outputs from the combiner module are coupled to the microscopes by two sets of four single-mode optic fibers in order to allow for the optimization of the TIRF angle for each wavelength independently. The emission is split into two or four spectral channels to allow for the simultaneous detection of up to four different fluorophores of wide selection and using many possible excitation and photoactivation schemes. We demonstrate the performance of this new setup by conducting two-color alternating excitation single-molecule fluorescence resonance energy transfer (FRET) and a technically challenging four-color FRET experiments on doubly labeled duplex DNA and quadruple-labeled Holliday junction, respectively.

Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins.

What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back-and-forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole-organizing protein PopZ, compensate for the defect in segregation regulation in ΔtipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self-organizing oscillator to create motion suitable for chromosome segregation.

Plasma-binding globulins and acute stress response.

Within studies of acute stress physiology an increase in glucocorticoid secretion is thought to be the primary mediator of tissue response to stress. Corticosteroid-binding globulin may regulate tissue availability of steroids, but has not been considered a dynamic component of the acute stress response. Here, we examined CBG level over the common 60-minute time frame in an acute capture and handling protocol to investigate whether CBG capacity is dynamic or static over short stressors. Using a comparative approach, we measured CBG response to capture and handling stress in nine species of birds, representing five orders and nine families. CBG capacity significantly declined within 30 - 60 minutes of capture in five of the nine species examined. This decline may serve to significantly increase the level of corticosterone reaching tissues during acute stress.

A landmark protein essential for establishing and perpetuating the polarity of a bacterial cell.

Polarity is often an intrinsic property of the cell, yet little is known about its origin or its maintenance over generations. Here we identify a landmark protein, TipN, which acts as a spatial and temporal cue for setting up the correct polarity in the bacterium Caulobacter crescentus. TipN marks the new pole throughout most of the cell cycle, and its relocation to the nascent poles at the end of division provides a preexisting reference point for orienting the polarity axis in the progeny. Deletion of tipN causes pleiotropic polarity defects, including frequently reversed asymmetry in progeny size and mislocalization of proteins and organelles. Ectopic localization of TipN along the lateral side of the cell creates new axes of polarity leading to cell branching and formation of competent cell poles. Localization defects of the actin-like protein MreB in the DeltatipN mutant suggest that TipN is upstream of MreB in regulating cell polarity.

Sustained benefits of growth hormone on body composition, fat utilization, physical strength and agility, and growth in Prader-Willi syndrome are dose-dependent.

BACKGROUND: Obesity and hypotonia in children with Prader-Willi syndrome (PWS) are accompanied by abnormal body composition resembling a growth hormone (GH) deficient state. Hypothalamic dysfunction in PWS includes decreased GH secretion, suggesting a possible therapeutic role for GH treatment. While recent studies have demonstrated short-term benefits of treatment with GH, a critical question is whether beneficial changes persist or wane with prolonged therapy, and whether these effects on body composition are dose-dependent as seen in adult GH deficiency. OBJECTIVES AND METHODS: After 24 months of GH theapy at a dose of 1 mg/m2/day ("standard dose"), the effects of 12 additional months of GH treatment at varying doses (0.3-1.5 mg/m2/day) on growth, body composition, strength and agility, pulmonary function, resting energy expenditure (REE), and fat utilization were assessed in 46 children with PWS. Percent body fat, lean muscle mass, and bone mineral density (BMD) were measured by dual X-ray absorptiometry (DXA). Indirect calorimetry was used to determine REE and to calculate respiratory quotient (RQ). RESULTS: During months 24-36 of GH therapy, further changes in body composition (decrease in fat mass, and increase in lean body mass), growth velocity, and REE occurred with standard and higher-dose GH therapy (1.5 mg/m2/day), but not with lower dose GH (0.3 mg/m2/day). Prior improvements in BMD, and strength and agility, which occurred during the initial 24 months, were sustained during the additional 12 months (to 36 months) regardless of dose. CONCLUSIONS: Salutary and sustained GH-induced changes in growth, body composition, and physical function in children with PWS require GH doses of >0.3 mg/m2/day. Conversely, BMD increased during the additional 12 months of therapy regardless of GH dose. Lower doses of GH, effective in improving body composition in adults with GHD, do not appear to be effective in children with PWS.

Patency following successful thrombolysis of occluded vascular grafts.

AIM: to determine patency after successful lysis of occluded bypass grafts. METHODS: data were collected from four centres with a wide experience of thrombolysis. Outcome following successful lysis was determined from prospectively collected data or case notes. Data from 75 patients, 53 men, were analysed. RESULTS: median age at time of lysis was 68 years (range 33-88). Median age of graft was 12 months (range 1-120). Patency at 12 months was 33% (95% conf. interval: 21-44%). There were no differences in patency depending on whether the graft was above or below the inguinal ligament or whether an additional procedure eg. percutaneous or vein patch angioplasty was carried out. However in those 48 cases when lysis was deemed complete, i.e. there was restoration of graft patency and at least one vessel run off patency at 12 months was 39% compared with 17% if lysis was incomplete (p=0.04). CONCLUSIONS: at the present time it is difficult to justify routine thrombolysis of occluded grafts when patency, based on intention to treat, is approximately 20% at one year. Following successful graft lysis the role of anticoagulation and careful graft surveillance require further investigation.

Causes of re-recurrence after polytetrafluoroethylene patch saphenoplasty for recurrent varicose veins.

BACKGROUND: The aim of this study was to determine whether a polytetrafluoroethylene (PTFE) patch sutured over the religated saphenofemoral junction could reduce the rate of recurrence after operation for recurrent varicose veins. METHODS: Fifty patients who had surgery for recurrent long saphenous incompetence (81 legs had a small PTFE patch sutured over the religated saphenofemoral junction. There were no major complications following surgery. Three patients had a wound infection or delayed healing. All patients were invited for clinical examination and duplex imaging at a median of 19 (range 6-39) months after operation. RESULTS: Some 38 of 43 patients (70 legs) remained satisfied with the results of surgery; 16 (23 per cent) of 70 legs had visible veins on inspection and eight of these (11 per cent) involved symptomatic recurrence. Duplex imaging showed that recurrence was due to saphenofemoral junction incompetence in ten legs; two appeared to have a major groin connection but the other eight appeared to have neovascularization. Other causes were thigh perforator reflux (three legs) and cross-groin collaterals (three). Eleven of the 16 legs with recurrence had varicography but in two the procedure was a technical failure. Two legs had evidence of a significant connection (more than 3 mm) and two a minor connection (less than 3 mm) to the femoral vein at the level of the PTFE patch, but in the remainder recurrence was due to upper thigh perforating veins. There was good concordance between duplex imaging and varicography. CONCLUSION: PTFE patch saphenoplasty appears to be safe. Although these are early results, the technique seems potentially as effective as other barrier methods that have been investigated; in ten legs (12 per cent) recurrence was attributed to failure at the level of the PTFE patch.

Sustained benefit after 2 years of growth hormone on body composition, fat utilization, physical strength and agility, and growth in Prader-Willi syndrome.

BACKGROUND: Obesity and hypotonia in children with Prader-Willi syndrome (PWS) are accompanied by abnormal body composition resembling a growth hormone (GH)-deficient state. Hypothalamic dysfunction in PWS includes decreased GH secretion, suggesting a possible therapeutic role for GH treatment. Although recent studies have demonstrated short-term benefits of treatment with GH, a critical question is whether beneficial changes persist or wane with prolonged therapy. OBJECTIVES AND METHODS: Effects of 24 months of GH treatment (1 mg/m(2)/d) on growth, body composition, strength and agility, pulmonary function, resting energy expenditure, and fat utilization were assessed in 35 children with PWS. Percent body fat, lean muscle mass, and bone mineral density were measured by dual-energy x-ray absorptiometry. Indirect calorimetry was used to determine resting energy expenditure and to calculate the respiratory quotient. RESULTS: Compared with baseline evaluations, increased height velocity (SD score -1.1 +/- 2.5 to 2.2 +/- 2.3; P <. 001), reduced percent body fat (46.4% +/- 8.4% to 40.3% +/- 10.0%, P <.001), and improved respiratory muscle function and physical strength and agility (sit-ups, weight-lifts, running speed, and broad jump; P <.01) were observed after 24 months of GH treatment. A decline in respiratory quotient (0.81 +/- 0.07 to 0.75 +/- 0.06; P <. 01) and a trend toward increased resting energy expenditure were also observed. Changes in response to GH occurred predominantly during the initial 12 months of GH therapy. CONCLUSIONS: Children with PWS had sustained increases in lean body mass, decreases in percent body fat, improvements in physical strength and agility, and increased fat oxidation after 24 months of GH therapy. However, between 12 and 24 months, the growth rate slowed. Consequently, encouraging initial results require even more prolonged study to draw conclusions regarding the long-term value of GH therapy in changing body composition in children with PWS.

Leptin concentrations in Prader-Willi syndrome before and after growth hormone replacement.

OBJECTIVE: This study explored leptin concentrations in Prader-Willi syndrome (PWS), a genetic disorder characterized by significant obesity and presumed hypothalamic dysfunction. The potential interaction of leptin metabolism with the growth hormone (GH) axis was also studied. STUDY DESIGN: Plasma leptin concentrations and percent body fat were determined by radioimmunoassay and dual energy x-ray absorptionmetry, respectively, in 23 children with Prader-Willi syndrome and 23 children with exogenous obesity. RESULTS: Log plasma leptin concentrations were positively correlated with percentage body fat in PWS (r = 0.844) and exogenous obesity (r = 0.869). When the regression lines for the two groups were compared, there were no differences in their slopes (P = 0.737) or intercepts (P = 0.701). Administration of recombinant human growth hormone to PWS children for 12 months significantly reduced both percentage body fat and plasma leptin concentrations, but the relationship of log plasma leptin to percentage body fat was unchanged. CONCLUSION: Prader-Willi syndrome is not accompanied by deranged leptin concentrations and there was no evidence of an interaction of the GH axis with leptin metabolism in these GH-deficient children.

Early ear problems and developmental problems at school age.

Retrospective history of middle ear disease was compared with developmental diagnosis in 507 consecutively referred school-age children. History of major ear problems was positively associated with discrepancies between the performance and verbal IQ on the WISC-R. History of major ear problems was positively associated with the presence of articulation disorders for children in the low social class, hyperactivity in the middle social class, and language problems in the high social class. A history of significant middle ear disease in early childhood should raise concerns for articulation difficulties and possible language problems in children presenting to clinicians with school problems.