Computational reverse-engineering of a spider-venom derived peptide active against Plasmodium falciparum SUB1

Background

Psalmopeotoxin I (PcFK1), a protein of 33 aminoacids derived from the venom of the spider Psalmopoeus Cambridgei, is able to inhibit the growth of Plasmodium falciparum malaria parasites with an IC in the low micromolar range. PcFK1 was proposed to act as an ion channel inhibitor, although experimental validation of this mechanism is lacking. The surface loops of PcFK1 have some sequence similarity with the parasite protein sequences cleaved by PfSUB1, a subtilisin-like protease essential for egress of Plasmodium falciparum merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity.

Findings

Molecular modeling and docking calculations showed that one loop could interact with the binding site of PfSUB1. The calculated free energy of binding averaged −5.01 kcal/mol, corresponding to a predicted low-medium micromolar constant of inhibition. PcFK1 inhibited the enzymatic activity of the recombinant PfSUB1 enzyme and the in vitro P.falciparum culture in a range compatible with our bioinformatics analysis. Using contact analysis and free energy decomposition we propose that residues A14 and Q15 are important in the interaction with PfSUB1.

Conclusions

Our computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity. This outlines the usefulness of advanced bioinformatics tools to predict the function of a protein structure. The structural features of PcFK1 represent an interesting protein scaffold for future protein engineering.     

Constitutive patterns of gene expression regulated by RNA-binding proteins

Background

RNA-binding proteins regulate a number of cellular processes, including synthesis, folding, translocation, assembly and clearance of RNAs. Recent studies have reported that an unexpectedly large number of proteins are able to interact with RNA, but the partners of many RNA-binding proteins are still uncharacterized.

Results

We combined prediction of ribonucleoprotein interactions, based on catRAPID calculations, with analysis of protein and RNA expression profiles from human tissues. We found strong interaction propensities for both positively and negatively correlated expression patterns. Our integration of in silico and ex vivo data unraveled two major types of protein–RNA interactions, with positively correlated patterns related to cell cycle control and negatively correlated patterns related to survival, growth and differentiation. To facilitate the investigation of protein–RNA interactions and expression networks, we developed the catRAPID express web server.

Conclusions

Our analysis sheds light on the role of RNA-binding proteins in regulating proliferation and differentiation processes, and we provide a data exploration tool to aid future experimental studies.     

Immunisation with recombinant PfEMP1 domains elicits functional rosette-inhibiting and phagocytosis-inducing antibodies to Plasmodium falciparum

Background

Rosetting is a Plasmodium falciparum virulence factor implicated in the pathogenesis of life-threatening malaria. Rosetting occurs when parasite–derived P. falciparum Erythrocyte Membrane Protein One (PfEMP1) on the surface of infected erythrocytes binds to human receptors on uninfected erythrocytes. PfEMP1 is a possible target for a vaccine to induce antibodies to inhibit rosetting and prevent severe malaria.

Methodology/Findings

We examined the vaccine potential of the six extracellular domains of a rosette-mediating PfEMP1 variant (ITvar9/R29var1 from the R29 parasite strain) by immunizing rabbits with recombinant proteins expressed in E. coli. Antibodies raised to each domain were tested for surface fluorescence with live infected erythrocytes, rosette inhibition and phagocytosis-induction. Antibodies to all PfEMP1 domains recognized the surface of live infected erythrocytes down to low concentrations (0.02–1.56 µg/ml of total IgG). Antibodies to all PfEMP1 domains except for the second Duffy-Binding-Like region inhibited rosetting (50% inhibitory concentration 0.04–4 µg/ml) and were able to opsonize and induce phagocytosis of infected erythrocytes at low concentrations (1.56–6.25 µg/ml). Antibodies to the N-terminal region (NTS-DBL1α) were the most effective in all assays. All antibodies were specific for the R29 parasite strain, and showed no functional activity against five other rosetting strains.

Conclusions/Significance

These results are encouraging for vaccine development as they show that potent antibodies can be generated to recombinant PfEMP1 domains that will inhibit rosetting and induce phagocytosis of infected erythrocytes. However, further work is needed on rosetting mechanisms and cross-reactivity in field isolates to define a set of PfEMP1 variants that could induce functional antibodies against a broad range of P. falciparum rosetting parasites.     

Plasmodium falciparum FIKK Kinase Members Target Distinct Components of the Erythrocyte Membrane

Background

Modulation of infected host cells by intracellular pathogens is a prerequisite for successful establishment of infection. In the human malaria parasite Plasmodium falciparum, potential candidates for erythrocyte remodelling include the apicomplexan-specific FIKK kinase family (20 members), several of which have been demonstrated to be transported into the erythrocyte cytoplasm via Maurer''s clefts.

Methodology

In the current work, we have knocked out two members of this gene family (Pf fikk7.1 and Pf fikk12), whose products are localized at the inner face of the erythrocyte membrane. Both mutant parasite lines were viable and erythrocytes infected with these parasites showed no detectable alteration in their ability to adhere in vitro to endothelial receptors such as chondroitin sulfate A and CD36. However, we observed sizeable decreases in the rigidity of infected erythrocytes in both knockout lines. Mutant parasites were further analyzed using a phospho-proteomic approach, which revealed distinct phosphorylation profiles in ghost preparations of infected erythrocytes. Knockout parasites showed a significant reduction in the level of phosphorylation of a protein of approximately 80 kDa for FIKK12-KO in trophozoite stage and a large protein of about 300 kDa for FIKK7.1-KO in schizont stage.

Conclusions

Our results suggest that FIKK members phosphorylate different membrane skeleton proteins of the infected erythrocyte in a stage-specific manner, inducing alterations in the mechanical properties of the parasite-infected red blood cell. This suggests that these host cell modifications may contribute to the parasites'' survival in the circulation of the human host.     

Plasmodium falciparum antigens on the surface of the gametocyte-infected erythrocyte

Background

The asexual blood stages of the human malaria parasite Plasmodium falciparum produce highly immunogenic polymorphic antigens that are expressed on the surface of the host cell. In contrast, few studies have examined the surface of the gametocyte-infected erythrocyte.

Methodology/Principal Findings

We used flow cytometry to detect antibodies recognising the surface of live cultured erythrocytes infected with gametocytes of P. falciparum strain 3D7 in the plasma of 200 Gambian children. The majority of children had been identified as carrying gametocytes after treatment for malaria, and each donated blood for mosquito-feeding experiments. None of the plasma recognised the surface of erythrocytes infected with developmental stages of gametocytes (I–IV), but 66 of 194 (34.0%) plasma contained IgG that recognised the surface of erythrocytes infected with mature (stage V) gametocytes. Thirty-four (17.0%) of 200 plasma tested recognised erythrocytes infected with trophozoites and schizonts, but there was no association with recognition of the surface of gametocyte-infected erythrocytes (odds ratio 1.08, 95% C.I. 0.434–2.57; P = 0.851). Plasma antibodies with the ability to recognise gametocyte surface antigens (GSA) were associated with the presence of antibodies that recognise the gamete antigen Pfs 230, but not Pfs48/45. Antibodies recognising GSA were associated with donors having lower gametocyte densities 4 weeks after antimalarial treatment.

Conclusions/Significance

We provide evidence that GSA are distinct from antigens detected on the surface of asexual 3D7 parasites. Our findings suggest a novel strategy for the development of transmission-blocking vaccines.     

Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein

Background

The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.

Methodology/Principal Findings

Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α_vβ₃ and α_vβ₅ integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α₅β₁ and α_iibβ₃. In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α_vβ₃ and α_vβ₅ integrins. A ⁶⁴Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3–5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.

Conclusions/Significance

We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.     

Declining responsiveness of Plasmodium falciparum infections to artemisinin-based combination treatments on the Kenyan coast

Background

The emergence of artemisinin-resistant P. falciparum malaria in South-East Asia highlights the need for continued global surveillance of the efficacy of artemisinin-based combination therapies.

Methods

On the Kenyan coast we studied the treatment responses in 474 children 6–59 months old with uncomplicated P. falciparum malaria in a randomized controlled trial of dihydroartemisinin-piperaquine vs. artemether-lumefantrine from 2005 to 2008. (ISRCTN88705995)

Results

The proportion of patients with residual parasitemia on day 1 rose from 55% in 2005–2006 to 87% in 2007–2008 (odds ratio, 5.4, 95%CI, 2.7–11.1; P<0.001) and from 81% to 95% (OR, 4.1, 95%CI, 1.7–9.9; P = 0.002) in the DHA-PPQ and AM-LM groups, respectively. In parallel, Kaplan-Meier estimated risks of apparent recrudescent infection by day 84 increased from 7% to 14% (P = 0.1) and from 6% to 15% (P = 0.05) with DHA-PPQ and AM-LM, respectively. Coinciding with decreasing transmission in the study area, clinical tolerance to parasitemia (defined as absence of fever) declined between 2005–2006 and 2007–2008 (OR body temperature >37.5°C, 2.8, 1.9–4.1; P<0.001). Neither in vitro sensitivity of parasites to DHA nor levels of antibodies against parasite extract accounted for parasite clearance rates or changes thereof.

Conclusions

The significant, albeit small, decline through time of parasitological response rates to treatment with ACTs may be due to the emergence of parasites with reduced drug sensitivity, to the coincident reduction in population-level clinical immunity, or both. Maintaining the efficacy of artemisinin-based therapy in Africa would benefit from a better understanding of the mechanisms underlying reduced parasite clearance rates.

Trial Registration

Controlled-Trials.com ISRCTN88705995     

Plasmodium falciparum malaria challenge by the bite of aseptic Anopheles stephensi mosquitoes: results of a randomized infectivity trial

Background

Experimental infection of malaria-naïve volunteers by the bite of Plasmodium falciparum-infected mosquitoes is a preferred means to test the protective effect of malaria vaccines and drugs. The standard model relies on the bite of five infected mosquitoes to induce malaria. We examined the efficacy of malaria transmission using mosquitoes raised aseptically in compliance with current Good Manufacturing Practices (cGMPs).

Methods and Findings

Eighteen adults aged 18–40 years were randomized to receive 1, 3 or 5 bites of Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of P. falciparum.Seventeen participants developed malaria; fourteen occurring on Day 11. The mean prepatent period was 10.9 days (9–12 days). The geometric mean parasitemia was 15.7 parasites/µL (range: 4–70) by microscopy. Polymerase chain reaction (PCR) detected parasites 3.1 (range: 0–4) days prior to microscopy. The geometric mean sporozoite load was 16,753 sporozoites per infected mosquito (range: 1,000–57,500). A 1-bite participant withdrew from the study on Day 13 post-challenge and was PCR and smear negative.

Conclusions

The use of aseptic, cGMP-compliant P. falciparum-infected mosquitoes is safe, is associated with a precise prepatent period compared to the standard model and appears more efficient than the standard approach, as it led to infection in 100% (6/6) of volunteers exposed to three mosquito bites and 83% (5/6) of volunteers exposed to one mosquito bite.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00744133","term_id":"NCT00744133"}}NCT00744133     

Safety and efficacy of the 10-day melarsoprol schedule for the treatment of second stage rhodesiense sleeping sickness

Objective

Assessment of the safety and efficacy of a 10-day melarsoprol schedule in second stage T.b. rhodesiense patients and the effect of suramin-pretreatment on the incidence of encephalopathic syndrome (ES) during melarsoprol therapy.

Design

Sequential conduct of a proof-of-concept trial (n = 60) and a utilization study (n = 78) using historic controls as comparator.

Setting

Two trial centres in the T.b. rhodesiense endemic regions of Tanzania and Uganda. Participants: Consenting patients with confirmed second stage disease and a minimum age of 6 years were eligible for participation. Unconscious and pregnant patients were excluded.

Main Outcome Measures

The primary outcome measures were safety and efficacy at end of treatment. The secondary outcome measure was efficacy during follow-up after 3, 6 and 12 months.

Results

The incidence of ES in the trial population was 11.2% (CI 5–17%) and 13% (CI 9–17%) in the historic data. The respective case fatality rates were 8.4% (CI 3–13.8%) and 9.3% (CI 6–12.6%). All patients discharged alive were free of parasites at end of treatment. Twelve months after discharge, 96% of patients were clinically cured. The mean hospitalization time was reduced from 29 to 13 days (p<0.0001) per patient.

Conclusions

The 10-day melarsoprol schedule does not expose patients to a higher risk of ES or death than does treatment according to national schedules in current use. The efficacy of the 10-day melarsoprol schedule was highly satisfactory. No benefit could be attributed to the suramin pre-treatment.

Trial Registration

Current Controlled Trials ISRCTN40537886     

Associations between Antibodies to a Panel of Plasmodium falciparum Specific Antigens and Response to Sub-Optimal Antimalarial Therapy in Kampala,Uganda

Background

Antibodies are important in the control of blood stage Plasmodium falciparum infection. It is unclear which antibody responses are responsible for, or even associated with protection, partly due to confounding by heterogeneous exposure. Assessment of response to partially effective antimalarial therapy, which requires the host to assist in clearing parasites, offers an opportunity to measure protection independent of exposure.

Methods

A cohort of children aged 1–10 years in Kampala, Uganda were treated with amodiaquine+sulfadoxine-pyrimethamine for uncomplicated malaria. Serum samples from the time of malaria diagnosis and 14 days later were analyzed for total IgG to 8 P. falciparum antigens using a quantitative indirect ELISA. Associations between antibody levels and risk of treatment failure were estimated using Cox proportional hazard regression.

Results

Higher levels of antibodies to apical membrane antigen 1 (AMA-1), but to none of the other 7 antigens were significantly associated with protection against treatment failure (HR 0.57 per 10-fold increase in antibody level, CI 0.41–0.79, p = 0.001). Protection increased consistently across the entire range of antibody levels.

Conclusions

Measurement of antibody levels to AMA-1 at the time of malaria may offer a quantitative biomarker of blood stage immunity to P. falciparum, a tool which is currently lacking.     

Latent Infection with Leishmania donovani in Highly Endemic Villages in Bihar,India

Introduction

Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.

Methods

We selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.

Results

We enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).

Discussion

Although infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.     

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria

Background

Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood.

Methods and Findings

Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8⁺ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4⁺ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs.

Conclusions

CD8⁺ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.     

Toxoplasma gondii chromodomain protein 1 binds to heterochromatin and colocalises with centromeres and telomeres at the nuclear periphery

Background

Apicomplexan parasites are responsible for some of the most deadly parasitic diseases afflicting humans, including malaria and toxoplasmosis. These obligate intracellular parasites exhibit a complex life cycle and a coordinated cell cycle-dependant expression program. Their cell division is a coordinated multistep process. How this complex mechanism is organised remains poorly understood.

Methods and Findings

In this study, we provide evidence for a link between heterochromatin, cell division and the compartmentalisation of the nucleus in Toxoplasma gondii. We characterised a T. gondii chromodomain containing protein (named TgChromo1) that specifically binds to heterochromatin. Using ChIP-on-chip on a genome-wide scale, we report TgChromo1 enrichment at the peri-centromeric chromatin. In addition, we demonstrate that TgChromo1 is cell-cycle regulated and co-localised with markers of the centrocone. Through the loci-specific FISH technique for T. gondii, we confirmed that TgChromo1 occupies the same nuclear localisation as the peri-centromeric sequences.

Conclusion

We propose that TgChromo1 may play a role in the sequestration of chromosomes at the nuclear periphery and in the process of T. gondii cell division.     

Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies

Background

The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.

Methods and Findings

Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC₅₀ 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC₅₀ of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.

Conclusions

The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.     

Methylation State of the EDA Gene Promoter in Chinese X-Linked Hypohidrotic Ectodermal Dysplasia Carriers

Introduction

Hypodontia, hypohidrosis, sparse hair and characteristic faces are the main characters of X-linked hypohidrotic ectodermal dysplasia (XLHED) which is caused by genetic ectodysplasin A (EDA) deficiency. Heterozygous female carriers tend to have mild to moderate XLHED phenotype, even though 30% of them present no obvious symptom.

Methods

A large Chinese XLHED family was reported and the entire coding region and exon–intron boundaries of EDA gene were sequenced. To elucidate the mechanism for carriers’ tempered phenotype, we analyzed the methylation level on four sites of the promoter of EDA by the pyrosequencing system.

Results

A known frameshift mutation (c.573–574 insT) was found in this pedigree. Combined with the pedigrees we reported before, 120 samples comprised of 23 carrier females from 11 families and 97 healthy females were analyzed for the methylation state of EDA promoter. Within 95% confidence interval (CI), 18 (78.26%) carriers were hypermethylated at these 4 sites.

Conclusion

Chinese XLHED carriers often have a hypermethylated EDA promoter.     

Clag9 is not essential for PfEMP1 surface expression in non-cytoadherent Plasmodium falciparum parasites with a chromosome 9 deletion

Background

The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1.

Methodology/Principal Findings

Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36.

Conclusions/Significance

Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications.