Francisco Romero Pastrana

University Medical Center Groningen

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Jan Maarten van Dijl

University Medical Center Groningen

Girbe Buist

University Medical Center Groningen

Marleen van Oosten

University Medical Center Groningen

Kevin P. Francis

University of California, Los Angeles

Nicholas M. Bernthal

University of California, Los Angeles

Lloyd Miller

Johns Hopkins Medicine

Mafalda Bispo

University Medical Center Groningen

Marjolein Heuker

University of Groningen

Gooitzen M. van Dam

University Medical Center Groningen

Hedzer Hoekstra

University Medical Center Groningen

Cooperative Institutions

University of Groningen

University Medical Center Groningen

University of California, Los Angeles

Johns Hopkins University

Mexican Social Security Institute

Orthopaedic Hospital

Johns Hopkins Medicine

UCLA Health

FujiFilm VisualSonics (Canada)

École Polytechnique Fédérale de Lausanne

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Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis

Applied Microbiology and Biotechnology (2016)

Ganesh Ram R. Visweswaran Dorota Kurek Monika Szeliga Francisco Romero Pastrana Oscar P. Kuipers

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

Lysin

Autolysis (biology)

Lytic cycle

Prophage

Autolysin

10.1007/s00253-016-7822-z

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A Lactococcus lactis expression vector set with multiple affinity tags to facilitate isolation and direct labeling of heterologous secreted proteins

Applied Microbiology and Biotechnology (2017)

Francisco Romero Pastrana Jolanda Neef Jan Maarten van Dijl Girbe Buist

The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His6)-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling. Specifically, we present two new groups of vectors allowing N- or C-terminal provision of proteins with a Strep-tag II or AVI-tag. Vectors for AVI-tagging encode an additional His6-tag for protein purification. Another set of vectors allows removal of N-terminal Strep- or His6-tags from expressed proteins with the tobacco etch virus protease. Two possible applications of the developed vectors are presented. First, we show that Strep-tagged LytM of Staphylococcus aureus in the growth medium of L. lactis can be directly bound to microtiter plates coated with an affinity reagent and used for enzyme-linked immunosorbent assays. Second, we show that the AVI-tagged Sle1 protein from S. aureus produced in L. lactis can be directly biotinylated and fluorescently labeled. The fluorescently labeled Sle1 was successfully applied for S. aureus re-binding studies, allowing subcellular localization by fluorescence microscopy. In conclusion, we have developed a set of expression vectors that enhances the versatility of L. lactis as a system for production of proteins with tags that can be used for affinity purification and site-specific protein labeling.

10.1007/s00253-017-8524-x

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Long-lasting untreated Achiles tendon ruptures. Plasty by a modified Christensen-Silverskjiold technique, without removing scarring fibrous tissue

Acta Ortopédica Mexicana (2002)

José Antonio Enríquez Castro Antonio García Hernández Francisco Romero Pastrana Atanasio Lópéz Valero Adrián González Trujano

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Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins

Scientific Reports (2018)

Francisco Romero Pastrana Jolanda Neef Dennis G. A. M. Koedijk Douwe de Graaf José C. Duipmans

Human antibody responses to pathogens, like Staphylococcus aureus, are important indicators for in vivo expression and immunogenicity of particular bacterial components. Accordingly, comparing the antibody responses to S. aureus components may serve to predict their potential applicability as antigens for vaccination. The present study was aimed at assessing immunoglobulin G (IgG) responses elicited by non-covalently cell surface-bound proteins of S. aureus, which thus far received relatively little attention. To this end, we applied plasma samples from patients with the genetic blistering disease epidermolysis bullosa (EB) and healthy S. aureus carriers. Of note, wounds of EB patients are highly colonized with S. aureus and accordingly these patients are more seriously exposed to staphylococcal antigens than healthy individuals. Ten non-covalently cell surface-bound proteins of S. aureus, namely Atl, Eap, Efb, EMP, IsaA, LukG, LukH, SA0710, Sle1 and SsaA2, were selected by bioinformatics and biochemical approaches. These antigens were recombinantly expressed, purified and tested for specific IgG responses using human plasma. We show that high exposure of EB patients to S. aureus is mirrored by elevated IgG levels against all tested non-covalently cell wall-bound staphylococcal antigens. This implies that these S. aureus cell surface proteins are prime targets for the human immune system.

Protein A

10.1038/s41598-018-21724-z

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The Isolation and Characterization of Two Novel Zinc Finger Genes from the Smith-Magenis Syndrome Region at 17p11.2

Francisco Romero Pastrana

Isolation

Zinc finger nuclease

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ARRANGEMENT OF β TUBULIN IN BULL SPERMATOZOA DURING NUCLEI DECONDENSATION

Archives of Andrology (2004)

Eduardo Gómez-Conde Francisco Romero Pastrana Miguel Vargas N. M. Delgado Rogelio Reyes-Reyes

Eukaryotic cells have internal scaffolding of microtubules cytoskeleton that gives them their characteristic shapes. We analyzed by immuno-fluorescence the shift and distribution of tubuline during in vitro bull sperm nuclei swelling by the action of heparin-reduced glutathione physiological decondensing agents. Sperm tubulin display a pattern that shows tubulin fluorescence all over the head, leaving the acrosome tip devoid of tubulin. In the second stage we can observe that the acrosomal zone is practically devoid of fluorescence and a net of fluorescent microtubules that seems to be anchored in the basal plate in the postacrosomal region. It is also possible to observe green spots of tubulin fluorescence in the nucleus periphery, that might represent clusters of chromatin hub-like bodies and/or the microtubule organizing center (MTOC). In the third stage, practically all tubulin moves backwards to the basal plate in the neck region of the sperm nuclei remaining in only the green fluorescence spots in the periphery of the swollen sperm nuclei. The results allow us to assume that tubulin mechanism rearrangement is considered to be necessary for the normal fertilization process.

Microtubule organizing center

10.1080/01485010490474869

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Comparison of two fluorescent probes in preclinical non-invasive imaging and image-guided debridement surgery of Staphylococcal biofilm implant infections

Scientific Reports (2021)

Howard Y. Park Stephen D. Zoller Vishal Hegde William L. Sheppard Zachary D. C. Burke

Implant-associated infections are challenging to diagnose and treat. Fluorescent probes have been heralded as a technologic advancement that can improve our ability to non-invasively identify infecting organisms, as well as guide the inexact procedure of surgical debridement. This study's purpose was to compare two fluorescent probes for their ability to localize Staphylococcus aureus biofilm infections on spinal implants utilizing noninvasive optical imaging, then assessing the broader applicability of the more successful probe in other infection animal models. This was followed by real-time, fluorescence image-guided surgery to facilitate debridement of infected tissue. The two probe candidates, a labelled antibiotic that targets peptidoglycan (Vanco-800CW), and the other, a labelled antibody targeting the immunodominant Staphylococcal antigen A (1D9-680), were injected into mice with spine implant infections. Mice were then imaged noninvasively with near infrared fluorescent imaging at wavelengths corresponding to the two probe candidates. Both probes localized to the infection, with the 1D9-680 probe showing greater fidelity over time. The 1D9-680 probe was then tested in mouse models of shoulder implant and allograft infection, demonstrating its broader applicability. Finally, an image-guided surgery system which superimposes fluorescent signals over analog, real-time, tissue images was employed to facilitate debridement of fluorescent-labelled bacteria.

Debridement (dental)

Fluorescence-lifetime imaging microscopy

10.1038/s41598-020-78362-7

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Differential epitope recognition in the immunodominant staphylococcal antigen A of Staphylococcus aureus by mouse versus human IgG antibodies

Scientific Reports (2017)

Dennis G. A. M. Koedijk Francisco Romero Pastrana Hedzer Hoekstra Sanne van den Berg Jaap Willem Back

The immunodominant staphylococcal antigen A (IsaA) is a potential target for active or passive immunization against the important human pathogen Staphylococcus aureus. Consistent with this view, monoclonal antibodies against IsaA were previously shown to be protective against S. aureus infections in mouse models. Further, patients with the genetic blistering disease epidermolysis bullosa (EB) displayed high IsaA-specific IgG levels that could potentially be protective. Yet, mice actively immunized with IsaA were not protected against S. aureus infection. The present study was aimed at explaining these differences in IsaA-specific immune responses. By epitope mapping, we show that the protective human monoclonal antibody (humAb) 1D9 recognizes a conserved 62-residue N-terminal domain of IsaA. The same region of IsaA is recognized by IgGs in EB patient sera. Further, we show by immunofluorescence microscopy that this N-terminal IsaA domain is exposed on the S. aureus cell surface. In contrast to the humAb 1D9 and IgGs from EB patients, the non-protective IgGs from mice immunized with IsaA were shown to predominantly bind the C-terminal domain of IsaA. Altogether, these observations focus attention on the N-terminal region of IsaA as a potential target for future immunization against S. aureus.

Epitope mapping

Conformational epitope

Immunofluorescence

10.1038/s41598-017-08182-9

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A human monoclonal antibody that specifically binds and inhibits the staphylococcal complement inhibitor protein SCIN

Virulence (2017)

Hedzer Hoekstra Francisco Romero Pastrana Hendrik P. J. Bonarius Kok P. M. van Kessel Goffe S. Elsinga

Staphylococcus aureus is a serious public health burden causing a wide variety of infections. Earlier detection of such infections could result in faster and more directed therapies that also prevent resistance development. Human monoclonal antibodies (humAbs) are promising tools for diagnosis and therapy owing to their relatively straightforward synthesis, long history of safe clinical use and high target specificity. Here we show that the humAb 6D4, which was obtained from a random screen of B-cells producing antibodies that bind to whole cells of S. aureus, targets the staphylococcal complement inhibitor (SCIN). The epitope recognized by 6D4 was localized to residues 26 to 36 in the N-terminus of SCIN, which overlap with the active site. Accordingly, 6D4 can inhibit SCIN activity as demonstrated through the analysis of C3b deposition on S. aureus cells and complement-induced lysis of rabbit erythrocytes. Importantly, while SCIN is generally regarded as a secreted virulence factor, 6D4 allowed detection of strongly increased SCIN binding to S. aureus cells upon exposure to human serum, relating to the known binding of SCIN to C3 convertases deposited on the staphylococcal cell surface. Lastly, we show that labeling of humAb 6D4 with a near-infrared fluorophore allows one-step detection of SCIN-producing S. aureus cells. Together, our findings show that the newly described humAb 6D4 specifically recognizes S. aureus SCIN, which can potentially be used for detection of human serum-incubated S. aureus strains expressing SCIN.

10.1080/21505594.2017.1294297

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Potential targets for immunotherapy and infection imaging on the cell surface of Staphylococcus aureus

Francisco Romero Pastrana

De bacterie Staphylococcus aureus is een uiterst flexibele ziekteverwekker die zich thuis voelt op het menselijk lichaam. Helaas vertoont de S. aureus bacterie in toenemende mate resistentie tegen antibiotica, waardoor infecties moeilijk te genezen zijn. De MRSA variant van S. aureus is in dit opzicht zeer berucht. S. aureus is bovendien in staat om te groeien op medische implantaten, zoals kunstknieen en -heupen. Dergelijke implantaatinfecties zijn moeilijk te genezen en in veel gevallen moet het geinfecteerde implantaat vervangen worden. Dit is zeer belastend voor patienten. Om deze redenen is er behoefte aan nieuwe middelen om S. aureus infecties zo vroeg mogelijk te diagnostiseren en gericht te bestrijden. Het onderzoek beschreven in dit proefschrift had tot doel om humane antistoffen tegen het oppervlak van de S. aureus bacterie te karakteriseren en te onderzoeken of ze gebruikt kunnen worden voor het opsporen van infecties. De resultaten laten zien dat eiwitten op het celoppervlak van S. aureus een mogelijk doelwit zijn voor nieuwe immuuntherapie om mensen tegen infectie te beschermen. Tevens worden twee zogenaamde monoklonale antistoffen beschreven die wellicht gebruikt kunnen worden om S. aureus infecties op implantaten te detecteren met geavanceerde beeldvormingstechnieken, zoals PET-scanning. Samenvattend kan geconcludeerd worden, dat het beschreven onderzoek een belangrijke bijdrage levert voor de ontwikkeling van nieuwe methodes om S. aureus infecties vroegtijdig op te sporen en te bestrijden.

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