Microbial composition affects the functioning of estuarine sediments

Although microorganisms largely drive many ecosystem processes, the relationship between microbial composition and their functioning remains unclear. To tease apart the effects of composition and the environment directly, microbial composition must be manipulated and maintained, ideally in a natural ecosystem. In this study, we aimed to test whether variability in microbial composition affects functional processes in a field setting, by reciprocally transplanting riverbed sediments between low- and high-salinity locations along the Nonesuch River (Maine, USA). We placed the sediments into microbial ‘cages'' to prevent the migration of microorganisms, while allowing the sediments to experience the abiotic conditions of the surroundings. We performed two experiments, short- (1 week) and long-term (7 weeks) reciprocal transplants, after which we assayed a variety of functional processes in the cages. In both experiments, we examined the composition of bacteria generally (targeting the 16S rDNA gene) and sulfate-reducing bacteria (SRB) specifically (targeting the dsrAB gene) using terminal restriction fragment length polymorphism (T-RFLP). In the short-term experiment, sediment processes (CO₂ production, CH₄ flux, nitrification and enzyme activities) depended on both the sediment''s origin (reflecting differences in microbial composition between salt and freshwater sediments) and the surrounding environment. In the long-term experiment, general bacterial composition (but not SRB composition) shifted in response to their new environment, and this composition was significantly correlated with sediment functioning. Further, sediment origin had a diminished effect, relative to the short-term experiment, on sediment processes. Overall, this study provides direct evidence that microbial composition directly affects functional processes in these sediments.     

Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.     

Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

Epidemic patch models applied to pandemic influenza: Contact matrix, stochasticity, robustness of predictions

Due to the recent emergence of H5N1 virus, the modelling of pandemic influenza has become a relevant issue. Here we present an SEIR model formulated to simulate a possible outbreak in Italy, analysing its structure and, more generally, the effect of including specific details into a model. These details regard population heterogeneities, such as age and spatial distribution, as well as stochasticity, that regulates the epidemic dynamics when the number of infectives is low. We discuss and motivate the specific modelling choices made when building the model and investigate how the model details influence the predicted dynamics. Our analysis may help in deciding which elements of complexity are worth including in the design of a deterministic model for pandemic influenza, in a balance between, on the one hand, keeping the model computationally efficient and the number of parameters low and, on the other hand, maintaining the necessary realistic features.     

Combination of two separate binding domains defines stoichiometry between type III secretion system chaperone IpgC and translocator protein IpaB

Type III secretion systems (TTSSs) utilized by enteropathogenic bacteria require the presence of small, acidic virulence-associated chaperones for effective host cell infection. We adopted a combination of biochemical and cellular techniques to define the chaperone binding domains (CBDs) in the translocators IpaB and IpaC associated with the chaperone IpgC from Shigella flexneri. We identified a novel CBD in IpaB and furthermore precisely mapped the boundaries of the CBDs in both translocator proteins. In IpaC a single binding domain associates with IpgC. In IpaB, we show that the binding of the newly characterized CBD is essential in maintaining the ternary arrangement of chaperone-translocator complex. This hitherto unknown function is reflected in the co-crystal structure as well, with an IpgC dimer bound to an IpaB fragment comprising both CBDs. Moreover, in the absence of this novel CBD the IpaB/IpgC complex aggregates. This dual-recognition of a domain in the protein by the chaperone in facilitating the correct chaperone-substrate organization describes a new function for the TTSS associated chaperone-substrate complexes.     

Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies

Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.     

Crystal structure of PrgI-SipD: insight into a secretion competent state of the type three secretion system needle tip and its interaction with host ligands

Many infectious gram-negative bacteria, including Salmonella typhimurium, require a Type Three Secretion System (T3SS) to translocate virulence factors into host cells. The T3SS consists of a membrane protein complex and an extracellular needle together that form a continuous channel. Regulated secretion of virulence factors requires the presence of SipD at the T3SS needle tip in S. typhimurium. Here we report three-dimensional structures of individual SipD, SipD in fusion with the needle subunit PrgI, and of SipD:PrgI in complex with the bile salt, deoxycholate. Assembly of the complex involves major conformational changes in both SipD and PrgI. This rearrangement is mediated via a π bulge in the central SipD helix and is stabilized by conserved amino acids that may allow for specificity in the assembly and composition of the tip proteins. Five copies each of the needle subunit PrgI and SipD form the T3SS needle tip complex. Using surface plasmon resonance spectroscopy and crystal structure analysis we found that the T3SS needle tip complex binds deoxycholate with micromolar affinity via a cleft formed at the SipD:PrgI interface. In the structure-based three-dimensional model of the T3SS needle tip, the bound deoxycholate faces the host membrane. Recently, binding of SipD with bile salts present in the gut was shown to impede bacterial infection. Binding of bile salts to the SipD:PrgI interface in this particular arrangement may thus inhibit the T3SS function. The structures presented in this study provide insight into the open state of the T3SS needle tip. Our findings present the atomic details of the T3SS arrangement occurring at the pathogen-host interface.     

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro

Background

Riboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.

Results

Results indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.

Conclusion

Thus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.