Importance of protein phosphorylation in bacterial pathogenicity
Phosphorylation of serine, threonine and tyrosine residues is the major regulatory post-translation modification in bacteria, catalyzed by protein kinases and phosphatases. In bacteria, it is believed that protein phosphorylation is essential for regulation of infection processes, such as adhesion to the host, triggering and regulation of pathogenic functions, and biochemical implications.
The most frequent phosphorylated proteins in bacteria are enzymes related to carbon/protein/nucleotide metabolism and housekeeping proteins as helicases, chaperones, ribosomal proteins and aminoacyl-tRNA-synthetases.
However, several steps can difficult the phosphoproteoma study as the enrichment method, sensitive mass spectrometry, low stoichiometry, limited dynamic range of detection methods, complexity and quantification difficulties. Combination of IMAC and nano-LC have been used as methodology to separate and identify phosphopeptides. However, previous enrichment phosphopeptides methods are required, whereas titanium dioxide chromatography has been mostly used.
Tyrosine phosphorylation in Pseudomonas aeruginosa phosphoproteome seems to be related to pathogenicity. Although, many other findings supporting the contribution of tyrosine phosphorylation to bacterial pathogenicity. Moreover, multiple phosphorylation sites has been observed in bacteria under stress or with overloaded proteolityc systems.
Protein phosphorylation seems to be correlated to pathogenic potential of bacteria, which can help to develop new phosphorylation-targeted prodrugs to control bacterial infections.
Identification of immunogenic proteins due to S. aureus biofilm chronic infection
Presence of biofilms is associated to recurrent infections. To identify immunogenic proteins in a S. aureus biofilm infection the following approach was performed:
Protein extracts from S. aureus strain obtained from a patient with osteomyelitis, were separated by 2D electrophoresis. Rabbits were infected with this strain in order to produce osteomyelitis and sera was collected at different timepoints. A westernblot was also done, incubating nitrocellulose of protein extracts with convalescent-phase serum from rabbit infected with this strain. Immunogens were identified by MALDI-TOF, and results confirmed by microarrays.
More than 2o immunogens were identified in cell wall, membrane and cytoplasm. Most of these immunogens were up-regulated, although, biofilm infection can persist. Comparison of results to acute infections showed that most of the identified immunogens were distinct, suggesting that antigens from biofilms are different from the imunogens from planktonic S. aureus during acute infections.
Model systems to study biofilms
This review summarizes in vitro and in vivo models developed in order to study microbial biofilms.
In vitro models described here include:
– microtiter plate-based system (MTP): one of the most used biofilm model system since is cheaper and less labour-intensive ; a closed system without flow; the environmental conditions in the well changes during the experiment; it is ideal to screening purposes. Also include Calgary Biofilm Device (variation of MTP) ideal to determnine antibiotic susceptibility and the Biofilm ring test which contain inert paramagnetic beads, already used to study the kinetics of Staphylococcus biofilm formation.
– flow displacement biofilm model systems: open system which require larger volumes of reagents and is more labour-intensive; include 2 major groups, Continuous flow stirred tank reactor (CFSTR) and the Plug Flow reactor (PFR). CFSTR system has a feed rate and a removal rate identical allowing a perfect mixing. PFR only has one flow direction and consequently the environmental conditions through the reactor change. This type of system include Modified Robbins device (MRD) (often a homemade system but also commercially avalilable), flow cell device, “well-suited for real-time non-destructive microscopic analyses of biofilms”, the CDC biofilm reactor (has been used to study biofilm formation), drip flow reactors (previously used to asses reduction of S. epidermidis biofilm formation by bacteriophages), rotating disc reactor (used to study several aspects of Staphylococcal biofilms).
– cell-culture-based model systems: mainly used to mimic the in vivo conditions, however limitations as absence of commensal flora and cell-mediated immune response are present.
– microfluidic devices: control of environmental conditions; with low flow rates; mostly used to develop biofilms under physiologically specific conditions.
In vivo models here referred are:
– C. elegans model: nematode usually used to assess virulence and effect of a particular compound.
– vertebrate models: such as rat, mouse, rabbit, monkey, dog, etc. can be used to study biofilm formation on catheters, subcutaneous and intraperitoneal foreign body, etc. Infections can be done prior or post implantation, or using bacteria embedded in agar beads.
This review also enumerates several ways to assess biofilm formation.
First, a recover biofilm-grown cells and disaggregation from the surface must be done (using dipping, rinsing, scraping, sonication). To quantify and visualize biofilm growth, the number of culturable cells may be determine by plate count methods, the crystal violet staining may be used to determine total biomass, SYTO 9 to determine live and dead cells, resazurin staining to determine the number of viable cells, and dimethylmethylene blue to assess the amount of extracellular polymers in the biofilm matrix. Other biofilm aspects can be studied with flow-cell technology and confocal microscopy.
After this overview, it is important to keep that the selection of the model system can influence the results.
Protease activity over staphylococcal adhesion
Zymography technique allows to assess protease activity, including this method in degradomic area.
Biofilm proteases has been related to inhibition of biofilm formation, and consequently has been studied as a potential therapeutic agent, since it reduces bacteria adhesion to surfaces.
In this work, 5 proteases were used to evaluate their antimicrobial potential in S. epidermidis and S. aureus biofilms.
Methodology was based on protease addition during biofilm formation and in human cell invasion processes, followed by SDS-PAGE and zymography of surface proteins.
Only serratiopeptidase (metalloprotease) showed inhibition of biofilm formation in all tested strains. Compared to strains without protease treatment, some protein bands disappeared or were reduced, due to the presence of a protease during biofilm formation. The main differences were observed with serine proteases proteinase K and chymotrypsin which acted as strong biofilm inhibitors. Proteins affected by protease treatment were excised and identified by mass spectrometry. The ability of proteases to interfere with S. aureus capacity to adhere and invade HeLa cells was also tested, showing variable results according to selected protease.
Besides the potential effect of proteases over biofilm formation, the major conclusion of this work is that protease action over biofilm formation was strain-dependent, and not related to protease class.
Recent findings on bacterial persistence
Persisters are a subpopulation of cells, generally nongrowing, that tolerate high doses of antibiotics.
The existence of persisters has been associated to chronic infections since the therapy fails.
This review summarizes the new findings on persisters regarding antibiotic tolerance, chemical signaling and in vivo studies.
Antibiotic tolerance in persister cells has been linked to: genes from metabolic and biosynthetic pathways, and energy production; suppression of oxidative stress; and a starvation response in order to prevent cell death. A SOS response dependent of increased expression of tisB toxin, can also induce persistent state, as shown in E. coli. Overexpression of toxin-antitoxin modules have been implicated in persistence formation in E. coli and M. tuberculosis.
Although quorum sensing has not been directly related to persisters formation, bacterial signaling molecule produced as a consequence of nutrient limitation, has recently been implicated in persistence regulation. As chemical environment can lead to heterogeneous response at a single cell level, genetic mechanisms, as genetic variation and stochastic gene expression, can also contribute to biological heterogeneity. Stochasticity in gene expression has been suggested as an explanation for phenotypic variability in a small population.
An in vivo model to study bacterial persistence is still lacking, however, recently, was made some advances in this area, with a well succeeded mice model to E. coli biofilm simulating a urinary tract infection.
Still, the main challenges in persistence study remain the validation of in vitro studies and development of an anti-persister drug.
Considerations on RNA-seq data analysis
Due to the high number of reads produced through RNA-sequencing, software tools has been used to perform the analysis. RNA-seq is been widely used to compare gene expression between different samples conditions.
(1) High throughput RNA-seq generates millions of short reads, most of the times from pair end reads, reducing the multi-mapping problem.
(2) Reads are mapped to the genome or transcriptome, to give a greater coverage.
(3) Data is normalized. A list of genes associated with P-value and fold change allows an expression comparison between and within samples. RPKM (reads per kilobase of exon model per million mapped reads) is one of the most used methods for normalization, based on the library size and gene lenght.
(4) Statistical test between samples is carry out, including analyses of biological replicates.
(5) Biological classification can be performed “to have a picture” of changes in sets of genes, tested in Gene Onthology terms.
In this review is also discussed the differences between RNA-seq and microarrays, as main methods to gene expression analysis.
Membrane and Cell wall proteome from S. aureus
Proteomic approach is been used to study pathogenesis of multiple microorganisms. Proteins from membrane and cell wall are the most studied to try to understand the interactions between pathogens and host. However, hydrophobic characteristics, alkaline pI and transmembrane regions, make membrane and cell surface proteins hard to be solubilized and analyzed by two dimensional electrophoresis (2-DE).
This work intends to construct a reference map of proteins from membrane and cell wall from S. aureus. For that, the authors tested different cell lysis and solubilization methods, followed by 2-DE. Proteins were identified by MALDI-TOF.
The different cell lysis procedures included enzymatic method with lysostaphin, a boiling method with SDS, a treatment with LiCl and a membrane extraction kit.
Between these cell lysis methods, the one with more spots and reproducible results was lysis with lysostaphin, which cleaves the staphylococcal peptidoglycan.
Regarding the solubilization solution, the authors tested different combinations of detergents (CHAPS, ASB-14), chaotropes (urea, thiourea) and reducing agents (DTT). They found that a solubilization solution formed with 8M urea, 2M thiourea, 1% ASB-14 and 1% DTT was the best solubilization condition allowing a better resolution, recovery spots and a wide molecular range of proteins.
This work shows how important is to choose an appropriate lysis method as a solubilization solution once this procedures modify the protein pattern.
Viable but non-culturable bacteria in biofilms from catheters
Since catheters-associated biofilms may represent a reservoir for VBNC (viable but non-culturable) bacteria it is important to find an efficient technique which allow detection of VBNC in this medical devices.
Maki technique is the reference standard technique to evaluate catheter colonization, but false negatives can occur occasionally.
This study focused in Maki-negative biofilms from central venous catheters samples which were stained with live/dead.
Used methodology included sonication and vortexing of catheters, and inoculation on TSB for 72hours. 88,6% of samples didn’t grown and were incubated with live/dead and examined in epifluorescence microscope.
The results showed that 77% of samples had viable bacteria. It was extracted DNA from them and performed real-time PCR targeting 16S rDNA and Staphylococci genes. 86,6% of samples with viable bacteria were positive by real-time PCR.
Moreover, it was found a correlation between temperature ≥38ºC and the presence of VBNC, suggesting a role of VBNC in clinical symptoms.
Magnesium and Pseudomonas fluorescens biofilm formation
The effect of magnesium ions were evaluated as a potentially adhesion factor in Pseudomonas fluorescens biofilm formation. How the magnesium affect biofilm structure is not yet known, however, mutations on Mg2+ transport systems resulted in decreased swarming and biofilm formation in Aeromonas hydrophilia. It is known that electrostatic interactions contribute to biofilm cohesion. Some studies on how divalent cations as Mg2+ and Ca2+ influence biofilm formation were already done
The influence of different Mg2+ concentration was evaluated on biofilm growth (biofilm and attached cells), namely by cell counting (using DAPI staining) and biofilm structure (with confocal laser scanning microscopy).
Mg2+ concentration lead to an increased attached cells but hadn’t influenced planktonic cells growth. However, the impact of Mg2+ concentration is dynamic over time.
Regarding the biofilm structure, the biofilms presented heterogeneous colonization. Surface colonization and depth increased with increasing Mg2+ concentrations.
Despite magnesium has different effects on bacterial adhesion, this study showed that Mg2+ increased initial attachment and altered formation and biofilm structure in Pseudomonas fluorescens specie.
Accordingly to Dunne and Burd (1992), magnesium concentrations as 16mM significantly enhanced in vitro adhesion of S. epidermidis to plastic.
Song B, Leff LG (2006). Influence of magnesium ions on biofilm formation by Pseudomonas fluorescens. Microbial Res; 161(4):355-361
RNA-seq: a tool to study transcriptomes
RNA-seq has been replacing microarray technology. Besides transcriptomic analysis, RNA-seq can be used to identify transcriptional start sites, to define operons and ncRNA.
The methodology includes:
1. RNA extraction, using a method that not affect the sample and allow harvesting enough RNA;
2. DNAse treatment;
3. RNAr depletion, to increase RNAm;
4. Covertion of RNA to cDNA, using random hexamer primers (this step can be eliminated to perform direct RNA sequencing);
5. Second DNA strand synthesis, to maintain the directional information;
6. Sequencing using sequencing plataforms as 454 Sequencing, Illumina or SOLiD;
7. Sequence alignment of data.
The advantages of RNA-seq are improved sensitivity, higher resolution and more discriminatory, once doesn’t use hybridization to probes. Replicates are highly consistent but the costs is an important issue when a research group decide to use RNA-seq.
Biofilm dormancy 