Streptococcus pyogenes TrxSR Two-Component System Regulates Biofilm Production in Acidic Environments.

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes. In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M protein, consistent with the transcriptional regulation of emm, which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

The KbvR Regulator Contributes to Capsule Production, Outer Membrane Protein Biosynthesis, Antiphagocytosis, and Virulence in Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that mostly affects patients with weakened immune systems, but a few serotypes (especially K1 and K2) are highly invasive and result in systemic infection in healthy persons. The ability to evade and survive the components of the innate immune system is critical in infection. To investigate the role and mechanism of transcription regulator KP1_RS12260 (KbvR) in virulence and defense against the innate immune response, kbvR deletion mutant and complement strains were constructed. The in vivo animal infection assay and in vitro antiphagocytosis assay demonstrate K. pneumoniae KbvR is an important regulator that contributes to virulence and the defense against phagocytosis of macrophages. The transcriptome analysis and phenotype experiments demonstrated that deletion of kbvR decreased production of capsular polysaccharide (CPS) and biosynthesis of partly outer membrane proteins (OMPs). The findings suggest that KbvR is a global regulator that confers pathoadaptive phenotypes, which provide several implications for improving our understanding of the pathogenesis of K. pneumoniae.

Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviously, detecting reagents such as a specific antibody is essential for the development of immune-based detection methods for C. jejuni or C. coli. In this study, in silico techniques were used to design a chimeric recombinant antigen, named multiepitope antigen (MEA), for the production of specific polyclonal antibody. To design MEA polypeptide based on C. jejuni fibronectin-binding protein or CadF, four conserved and unique antigenic peptides were identified and fused together directly. The C. jejuni CadF-based MEA polypeptide fused with two single six-histidine tags at both C- and N-terminal ends was expressed under Escherichia coli expression system. The recombinant MEA was successfully produced and purified by Ni-NTA resin with a high satisfactory yield. Indirect ELISA results showed that anti-MEA polyclonal antibody derived from rabbit serum had a titer of 16,000, indicating high antigenicity of MEA polypeptide. Dot blot results also confirmed that the produced anti-MEA antibody could specifically recognize both C. jejuni and C. coli whole cells as expected while there was no cross-reactivity to non-Campylobacter spp. tested in this study.

Soluble Expression, One-Step Purification and Characterization of Recombinant Human Growth Hormone Fused with ompA3 in Escherichia coli.

BACKGROUND: Human growth hormone (hGH) is the first recombinant protein approved for the treatment of human growth hormone deficiency. However, expression in inclusion bodies and low expression levels are enormous challenges for heterologous expression of hGH in Escherichia coli. OBJECTIVE: To increase the soluble expression of recombinant hGH with correct folding in E. coli. METHODS: We constructed a new recombinant expression plasmid containing the coding sequence of the outer membrane protein A (ompA3) which was used for the expression in Transetta (DE3) E. coli. In order to simplify the purification process and cleavage of recombinant proteins, the fusion sequence should contain hexahistidine-tag (His6) and enterokinase recognition sites (D4K). The effect of different expression conditions on recombinant hGH expression was optimized in flask cultivations. Furthermore, the periplasmic solution containing soluble hGH was purified by Ni-NTA affinity chromatography. Circular dichroism (CD), western blot and mass spectrometry analyses were used to characterize the protein. Moreover, the growth-promoting effect of the purified hGH was also evaluated by cell proliferation assay. RESULTS: High-level expression (800 µg/mL) was achieved by induction with 0.5 mM IPTG at 30°C for 10 hours. The purity of hGH was over 90%. The immunological activity, secondary structure and molecular weight of the purified hGH were consistent with native hGH. The purified hGH was found to promote the growth of MC3T3-E1 cells, and was found to show the highest activity at a concentration of 100 ng/mL. CONCLUSION: Our research provides a feasible and convenient method for the soluble expression of recombinant hGH in E. coli, and may lay a foundation for the production and application of hGH in the industry.

Altered gene expression profile of Wolbachia pipientis wAlbB strain following transinfection from its native host Aedes albopictus to Aedes aegypti cells.

Wolbachia is an obligate intracellular bacterial symbiont prevalent among arthropods and nematodes. To survive and reproduce, Wolbachia interacts with and modifies host subcellular structures, while sensing and responding to changes within the cellular environment. In mutualistic associations, Wolbachia may provision the host with metabolites, or help to maintain the chemical homeostasis of the host cell. Some strains can rapidly invade insect populations by manipulating host reproductive biology, while also preventing viral replication, allowing their use in vector control of arthropod-borne viruses. The Aedes albopictus-derived strain wAlbB is promising in this regard. When transinfected into the Yellow fever mosquito, Aedes aegypti, wAlbB reaches high frequencies within wild populations, and strongly inhibits viral transmission. Despite its obvious potential, much is still unknown about the molecular interactions between Wolbachia and host that enable its use in vector control. Furthermore, most Wolbachia transinfection research to date has focused on host effects. In the current study, we used a cell line model to explore the effect of transinfection of wAlbB from Ae. albopictus to Ae. aegypti. Using RNA sequencing, we show that several genes associated with host-symbiont interactions were downregulated by transinfection, with the greatest downregulation exhibited by prophage-associated genes.

Investigation of the effects of oxidative stress-inducing factors on culturing and productivity of Bordetella pertussis.

The stress response of Bordetella pertussis during fermentation was assessed by means of fluorescence-based techniques. During the manufacturing of vaccines, B. pertussis is subjected to stress during adaptation to a new environment and operating conditions in the bioreactor, which can have harmful consequences on growth and protein yield. In this study, stress was imposed by varying the percentage of dissolved oxygen (DO) and inoculum size, and by adding rotenone and hydrogen peroxide. In this study, fluorescence spectroscopy is used as a tool for measuring oxidative stress. High levels of DO during fed-batch operation had no detrimental effect on growth, but the specific productivity of pertactin (PRN) decreased. Cultures that were started with an inoculum size that was 10 times smaller than the control resulted in significantly less PRN as compared to controls where reduction was more significant in flasks as compared to bioreactors. A comparison of filtered to heat-sterilized media revealed that filtered media offered a protective effect against H2 O2 . Heat sterilization of the media might result in the destruction of components that offer protection against oxidative stress. Nonetheless, filter sterilization on its own would be insufficient for large-scale manufacturing. It should be emphasized that the effects of these stressors while investigating for other microorganisms have not been studied for B. pertussis.

The big BAM theory: An open and closed case?

The ß-barrel assembly machinery (BAM) is responsible for the biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria. These OMPs have a membrane-embedded domain consisting of a ß-barrel fold which can vary from 8 to 36 ß-strands, with each serving a diverse role in the cell such as nutrient uptake and virulence. BAM was first identified nearly two decades ago, but only recently has the molecular structure of the full complex been reported. Together with many years of functional characterization, we have a significantly clearer depiction of BAM's structure, the intra-complex interactions, conformational changes that BAM may undergo during OMP biogenesis, and the role chaperones may play. But still, despite advances over the past two decades, the mechanism for BAM-mediated OMP biogenesis remains elusive. Over the years, several theories have been proposed that have varying degrees of support from the literature, but none has of yet been conclusive enough to be widely accepted as the sole mechanism. We will present a brief history of BAM, the recent work on the structures of BAM, and a critical analysis of the current theories for how it may function.

Soluble versions of outer membrane cytochromes function as exporters for heterologously produced cargo proteins.

This study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway.

Continuous production of Neisseria meningitidis outer membrane vesicles.

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.

Lipoproteins: Structure, Function, Biosynthesis.

The Lpp lipoprotein of Escherichia coli is the first identified protein with a covalently linked lipid. It is chemically bound by its C-terminus to murein (peptidoglycan) and inserts by the lipid at the N-terminus into the outer membrane. As the most abundant protein in E. coli (106 molecules per cell) it plays an important role for the integrity of the cell envelope. Lpp represents the type protein of a large variety of lipoproteins found in Gram-negative and Gram-positive bacteria and in archaea that have in common the lipid structure for anchoring the proteins to membranes but otherwise strongly vary in sequence, structure, and function. Predicted lipoproteins in known prokaryotic genomes comprise 2.7% of all proteins. Lipoproteins are modified by a unique phospholipid pathway and transferred from the cytoplasmic membrane into the outer membrane by a special system. They are involved in protein incorporation into the outer membrane, protein secretion across the cytoplasmic membrane, periplasm and outer membrane, signal transduction, conjugation, cell wall metabolism, antibiotic resistance, biofilm formation, and adhesion to host tissues. They are only found in bacteria and function as signal molecules for the innate immune system of vertebrates, where they cause inflammation and elicit innate and adaptive immune response through Toll-like receptors. This review discusses various aspects of Lpp and other lipoproteins of Gram-negative and Gram-positive bacteria and archaea.

Different Virulence Capabilities and ompA Expressions in ST2 and ST513 of Multidrug-Resistant Acinetobacter baumannii.

Successful clones of Acinetobacter baumannii cause a variety of nosocomial infections through serum resistance, biofilm formation, and antimicrobial resistance as virulence capabilities. Fifty clinical isolates of multidrug-resistant (MDR) A. baumannii were analyzed for clonal relatedness, serum resistance, biofilm formation, and in vivo assays. Furthermore, some virulence genes, sequence variation of ompA, and its expression were studied. The MLST (multilocus sequence typing) results showed that there were three sequence types among MDR isolates including ST2 (64%, 32/50), ST513 (30%, 15/50), and ST1 (6%, 3/50). The data showed that the clinical isolates recovered from sputum had mostly high biofilm-formation capacity, while isolates recovered from host interior fluids had high serum resistance. The results of PCR assays and in silico analysis represented patterns of virulence genes and even ompA sequence variations among MDR isolates which were clonally dependent. While quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that bacteremia-producing strains in C57/BL6 mice significantly overexpress ompA (P < 0.05) and have a direct relation with the level of IL-6 in bloodstream of mice. Moreover, the expressions of ompA among indistinguishable clones (ST2 or ST513) were clonally independent.

Accumulation of virulence-associated proteins in Campylobacter jejuni Outer Membrane Vesicles at human body temperature.

Campylobacter jejuni is the major cause of bacterial gastroenteritis in humans. In contrast, colonization in avian hosts is asymptomatic. Body temperature differs between human (37 °C) and avian (42 °C) hosts, and bacterial growth in 37 °C is therefore a potential cue for higher virulence properties during human infection. The proteome of the bacteria was previously shown to be altered by temperature. Here we investigated whether temperature has an effect on the C. jejuni outer membrane vesicle (OMV) proteome, as OMVs are considered to be bacterial vehicles for protein delivery and might play a role during infection. OMVs isolated from C. jejuni strain 81-176 grown at 37 °C and 42 °C were analyzed by LC-ESI-MS/MS. 181 proteins were detected in both sample groups, one protein was exclusively present, and three were absent in OMVs from 37 °C. Of the 181 proteins, 59 were differentially expressed; 30 proteins were detected with higher abundance, and 29 proteins with lower abundance at 37 °C. Among the more highly abundant proteins, significantly more proteins were predicted to be associated with virulence. These data show that temperature has an impact on the property of the OMVs, and this might affect the outcome of colonization/infection by C. jejuni in different hosts. SIGNIFICANCE: While C. jejuni is considered as a commensal bacterium in avian hosts, it causes symptomatic infection in humans. As the host body temperature is one differentiating factor, the growth temperature can potentially affect the virulence properties of the bacteria, but also OMVs that are released during growth. By using a proteomic approach, in this study, we observed that the protein content of OMVs isolated from C. jejuni is affected by growth temperature and that more proteins related to virulence are associated with OMVs at 37°C growth temperature. This property indicates that C. jejuni OMVs have a potential role for the outcome of human infection.

Predominant Shift of Different P44-Expressing Anaplasma phagocytophilum in Infected HL-60, THP-1, NB4, and RF/6A Cell Lines.

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium that dominantly produces P44 outer membrane proteins encoded by the p44/msp2 multigene family, which are major antigens for serodiagnosis. However, A. phagocytophilum antigens from cultures with different cell lines seem to have varying reactivities with sera. In this study, we performed RNA-seq to investigate the P44 expression of A. phagocytophilum propagated in 4 cell lines. In infected HL-60 cells, the P44-2b transcript was predominant in the first RNA-seq analysis (HL-60.1). However, the P44-23 transcript was predominant in the second RNA-seq analysis at 1 month after additional passages (HL-60.2). We further analyzed the P44 expression of A. phagocytophilum cultured in THP-1, NB4, and RF/6A cells through consecutive passages in the same cell lines for 1 year after transferring A. phagocytophilum from infected HL-60 cells to the respective cell lines. In the long-term cultures, P44-18, P44-78, and P44-51 were predominantly transcribed in infected THP-1, NB4, and RF/6A cells, respectively. Therefore, the predominant shifts of different P44-expressing transcripts of A. phagocytophilum might occur during cell culture even in the same cell line at different time points of sample harvest (HL-60.1 and HL-60.2), which may be attributed to host cell adaptation/selection/interaction.

Transcriptional regulation of the ferric aerobactin receptor gene by a GntR-like repressor IutR in Vibrio furnissii.

We found that Vibrio furnissii can utilize aerobactin (AERO) as a xenosiderophore. A homology search of its genome revealed that this bacterium possesses genes encoding an AERO-mediated iron acquisition system similar to that of V. vulnificus. The system consists of the ABC transporter gene vatCDB, the GntR-type transcriptional repressor gene iutR, and the outer membrane receptor gene iutA. The functions of the vatCDB operon and iutA in V. furnissii were confirmed by the inability of the corresponding deletion mutants to utilize AERO. Reverse transcription-quantitative PCR revealed that iutA transcription under iron-limiting conditions was extensively activated by the addition of AERO to the growth medium; therefore, we focused on elucidating this phenomenon. Electrophoretic mobility shift and DNase I footprinting assays revealed that glutathione S-transferase-fused IutR (GST-IutR) bound directly to a specific palindromic sequence in the iutA promoter region. However, GST-IutR did not bind to this sequence when either AERO or ferric AERO was present in the assay mixture. These in vitro findings suggest that, under iron-limiting conditions, iutA transcription in V. furnissii is artfully regulated both by IutR, acting as a direct repressor of iutA, and by AERO, acting as an effector for IutR, leading to the derepression of iutA transcription.

Mannheimia haemolytica OmpP2-like is an amyloid-like protein, forms filaments, takes part in cell adhesion and is part of biofilms.

Mannheimia haemolytica causes respiratory disease in cattle. Amyloid proteins are a major component of biofilms; they aid in adhesion and confer resistance against several environmental insults. The amyloid protein curli is highly resistant to protease digestion and physical and chemical denaturation and binds Congo red (CR) dye. The purpose of this study was to characterize an approximately 50-kDa CR-binding amyloid-like protein (ALP) expressed by M. haemolytica. This protein resisted boiling and formic acid digestion and was recognized by a polyclonal anti-Escherichia coli curli serum, suggesting its relationship with amyloid proteins. Immunolabeling and transmission electron microscopy showed that antibodies bound long, thin fibers attached to the bacterial surface. Mass spectrometry analysis indicated that these fibers are M. haemolytica OmpP2-like proteins. The purified protein formed filaments in vitro, and antiserum against it reacted positively with biofilms. An in silico analysis of its amino acid sequence indicated it has auto-aggregation properties and eight amyloid peptides. Rabbit polyclonal antibodies generated against this ALP diminished the adhesion of ATCC 31612 and BA1 M. haemolytica strains to A549 human epithelial cells, indicating its participation in cell adhesion. ALP expressed by M. haemolytica may be important in its pathogenicity and ability to form biofilms.

Formation of Sublethally Injured Yersinia enterocolitica, Escherichia coli O157:H7, and Salmonella enterica Serovar Enteritidis Cells after Neutral Electrolyzed Oxidizing Water Treatments.

The impact of neutral electrolyzed oxidizing (NEO) water treatments on the formation of sublethally injured Yersinia enterocolitica, Escherichia coli O157:H7, and Salmonella enterica serovar Enteritidis cells was evaluated. When pathogens were treated with 6% NEO water, approximately 38% of the treated Yersinia population and 25% of the treated Salmonella population became sublethally injured. The highest sublethally injured population was found when Salmonella cultures were treated with 3% NEO water. Regardless of the NEO water concentration used, no sublethally injured E. coli O157:H7 cells were found. To evaluate the sensitivity of NEO water-treated cells, four additional stresses (heat treatment, pH, NaCl, and bile salt) were tested. NEO water treatments did not generate any cross protection of treated cells against the other stresses. The diluted NEO water treatments in combination with heat treatment at 51°C for 10 min led to the best synergistic antimicrobial effects with a combined reduction of 7 logs. The gene expression results showed that NEO water treatments led to the upregulation of ompR, ail, and ycfR These genes are known for their involvement in cells' environmental stress responses. In summary, this study investigated the sublethal injury in pathogenic cells caused by NEO water treatments. Although sublethal injury was discovered, when combined with other mild stresses, the synergistic antimicrobial effects were able to further reduce the numbers of viable pathogenic cells. These results demonstrate the great application potential of NEO water as a nonthermal and less corrosive antimicrobial treatment.IMPORTANCE Neutral electrolyzed oxidizing (NEO) water is a nonthermal and less corrosive antimicrobial treatment that has been demonstrated to have efficacy in reducing microbial contamination in food, including meat, fresh fruit, and vegetables. However, NEO water treatments can cause sublethal injury to pathogenic cells, resulting in cells that retain their viability. Consequently, these sublethally injured pathogenic cells become a serious food safety concern. This study evaluated the formation of sublethally injured Yersinia enterocolitica, Escherichia coli O157:H7, and Salmonella enterica serovar Enteritidis cells by NEO water treatments and the potential cross protection against heat, pH, NaCl, or bile salt stresses that it may generate. No cross protection was observed. By combining NEO water treatments with sublethal levels of additional stresses, significant synergistic antimicrobial outcomes were achieved. These results indicate that mild processing treatments, when combined, can effectively reduce pathogen populations while minimizing the negative impacts on food quality.

Association of virulence gene expression with colistin-resistance in Acinetobacter baumannii: analysis of genotype, antimicrobial susceptibility, and biofilm formation.

BACKGROUND: Acinetobacter baumannii causes difficult-to-treat nosocomial infections, which often lead to morbidity due to the development of antimicrobial drug resistance and expression of virulence genes. Data regarding the association of resistance to colistin, a last treatment option, and the virulence gene expression of A. baumannii is scarce. METHODS: We evaluated the MLVA genotype, antimicrobial resistance, and biofilm formation of 100 A. baumannii isolates from burn patients, and further compared the in vitro and in vivo expression of four virulence genes among five colistin-resistant A. baumannii (Cst-R-AB) isolates. Five Cst-R-AB isolates were tested; one from the present study, and four isolated previously. RESULTS: Our results showed that reduced expression of recA, along with increased in vivo expression of lpsB, dnaK, and blsA; are associated with colistin resistance among Cst-R-AB isolates. Differences in virulence gene expressions among Cst-R-AB isolates, may in part explain common discrepant in vitro vs. in vivo susceptibility data during treatment of infections caused by Cst-R-AB. CONCLUSIONS: Our findings highlight the intricate relationship between colistin-resistance and virulence among A. baumannii isolates, and underscore the importance of examining the interactions between virulence and antimicrobial resistance toward efforts to control the spread of multidrug-resistant A. baumannii (MDR-AB) isolates, and also to reduce disease severity in burn patients with MDR-AB infection.

Estimation of the bacteriocin ColE7 conjugation-based "kill" - "anti-kill" antimicrobial system by real-time PCR, fluorescence staining and bioluminescence assays.

The efficiency of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system, was assessed using real-time PCR, flow cytometry and bioluminescence. The ColE7 antimicrobial system consists of the genetically modified Escherichia coli strain Nissle 1917 harbouring a conjugative plasmid (derivative of the F-plasmid) encoding the "kill" gene (ColE7 activity gene) and a chromosomally encoded "anti-kill" gene (ColE7 immunity gene). On the basis of traJ gene expression in the killer donor cells, our results showed that the efficiency of the here studied antimicrobial system against target E. coli was higher at 4 than at 24 h. Flow cytometry was used to indirectly estimate DNase activity of the antimicrobial system, as lysis of target E. coli cells in the conjugative mixture with the killer donor strain led to reduction in cell cytosol fluorescence. According to a lux assay, E. coli TG1 (pXen lux+ Apr ) with constitutive luminescence were killed already after 2 h of treatment. Target sensor E. coli C600 with DNA damage SOS-inducible luminescence showed significantly lower SOS induction 6 and 24 h following treatment with the killer donor strain. Our results thus showed that bioluminescent techniques are quick and suitable for estimation of the ColE7 bacterial conjugation-based antimicrobial system antibacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial antimicrobial resistance is worldwide rising and causing deaths of thousands of patients infected with multi-drug resistant bacterial strains. In addition, there is a lack of efficient alternative antimicrobial agents. The significance of our research is the use of a number of methods (real-time PCR, flow cytometry and bioluminescence-based technique) to assess the antibacterial activity of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system. Bioluminescent techniques proved to be rapid and suitable for estimation of antibacterial activity of ColE7 bacterial conjugation-based antimicrobial system and possibly other related systems.

Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis

Background: P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, which encodes for this protein, was cloned in Escherichia coli and the P64k recombinant protein was expressed in E. coli K12 GC366 cells under the control of a tryptophan promoter. P64k was expressed as an intracellular soluble protein about 28% of the total cellular protein. Several scale-up criteria of fermentation processes were studied to obtain the recombinant P64k protein at the pilot production scale. Results: The best operational conditions at a larger scale production of P64k recombinant protein were studied and compared using the four following criteria: Constant Reynold's number (Re constant), Constant impeller tip speed (n di constant), Constant power consumption per unit liquid volume (P/V constant) and Constant volumetric oxygen transfer coefficients (KLa/k constant). The highest production of the recombinant protein was achieved based on the constant KLa/k scale-up fermentation criterion, calculating the aeration rate (Q) and the impeller agitation speed (n) by iterative process, keeping constant the KLa/k value from bench scale. The P64k protein total production at the 50 l culture scale was 546 mg l -1 in comparison with the 284 mg l -1 obtained at 1.5 l bench scale. Conclusions: The methodology described herein, for the KLa/k scale-up fermentation criterion, allowed us to obtain the P64k protein at 50 l scale. A fermentation process for the production of P64k protein from N. meningitidis was established, a protein to be used in future vaccine formulations in humans.