Deconstructing export of malaria proteins.

The virulence of the malaria parasite Plasmodium falciparum is mediated by parasite proteins exported to the surface of infected erythrocytes. In this issue, Maier et al. (2008) report a screen of malaria parasite genes predicted to be involved in parasite protein export and trafficking within the host erythrocyte and discover that many more than expected are essential for parasite survival in vitro.

Persistent Cranial Defects After Endoscopic Sagittal Synostosis Surgery.

INTRODUCTION: Incomplete cranial ossification is a rare complication of calvarial-vault remodeling for sagittal synostosis often requiring reoperation. Studies show an incidence ranging from 0.5% to 18%. METHODS: Infants with sagittal synostosis who underwent endoscopic sagittal synostectomy and barrel stave osteotomies with postoperative orthotic helmeting between 2003 and 2021 were included with minimum follow-up until the completion of helmeting. RESULTS: Of 90 patients, 86 met inclusion; 3 had defects (3.5%). Patients with and without cranial defects had no difference in age of surgery (113 versus 131 d), duration helmeting (6.6 versus 7.0 mo), or perioperative/postoperative complications. Two underwent reoperation for recurrence. Patients with cranial defects manifested the evidence of developmental concerns more than patients without (100% versus 16.9%).The average cranial defect size was 19.33 cm 2 and age at surgery 4.29 years. All were managed with cranial particulate bone grafting with addition of bone matrix and SonicWeld plate. The first had 6×6 cm posterior defect requiring cranioplasty at 4.86 years with excellent healing. The second had a 3×6 cm posterior and 1×1 cm anterior defect, underwent cranioplasty at 4.14 years with persistent 4×6 defect, requiring repeat cranioplasty at 5.3 years. The third had a 3×5 cm posterior defect and underwent cranioplasty at 3.88 years with continued defect, planning for repeat intervention. CONCLUSIONS: This is the largest documented series of reoperations for incomplete ossification after endoscopic sagittal synostectomy with postoperative helmet treatment. The authors report a 3.5% rate of cranial defects, managed with bone grafting, bone matrix, and absorbable plates. Patients with poor ossification may have a propensity toward developmental concerns.

Endoscopic Spring-Mediated Distraction for Unilambdoid Craniosynostosis.

BACKGROUND: Craniosynostosis treatment modalities have changed over time. These have included open calvarial remodeling, suturectomy with helmet molding, hand-powered distraction devices, and spring-mediated distraction. Implantable springs were first described for their use in treatment of craniosynostosis in 1998 (Lauritzen et al, Plast Reconstr Surg 121;2008:545-554). They have been used for the correction of craniosynostosis involving single and multiple sutures and have been placed through both endoscopic and open approaches. Their use for correction of lambdoid synostosis has been previously only described using an open approach (Arnaud et al, Child Nerv Syst 28;2012:1545-1549). The senior author has performed spring-mediated distraction for treating unilambdoid craniosynostosis using an endoscopic approach, which is described below and has not previously been reported by other authors. METHODS: A retrospective analysis of our series of endoscopic unilambdoid synostosis repairs is included in this article. Patients were analyzed based on patient characteristics, operative details, and outcomes. The operation commences by approaching the lambdoid suture endoscopically through a 2- to 3-cm incision lateral to the lambdoid suture. Burr holes are placed on either side of the suture and a suturectomy is performed. Springs are bent preoperatively to a predetermined force. Two springs are placed across the suturectomy defect and the skin is closed. The patient is monitored for improvement in head shape and cranial X-rays are performed to measure the degree of distraction. RESULTS: Seven patients underwent endoscopic spring-mediated distraction for unilambdoid craniosynostosis. The average age at the time of operation was 9.4 months. The median force of each spring placed was 7.0 N. The median length of hospital stay after spring placement was 2 days. Springs were removed at 5.6 months on average. Five patients had X-rays immediately after placement and prior to removal. Each spring expanded an average distance of 15.3 mm. There were no surgical complications. Three patients had both preoperative and postoperative computed tomography scans available. The angle of the cranial base, calculated by comparing foramen magnum to cribriform plate angles, improved 5.8° (12.3 preoperatively to 6.6 postoperatively). CONCLUSION: Endoscopic spring-mediated distraction is a safe and effective method of treatment for unilambdoid craniosynostosis. The series represents the largest experience with this technique. The approach can be considered in all patients with unilambdoid synostosis given the efficacious improvement in vault remodeling, low patient morbidity, short operating time, and minimal inpatient stay.

Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites.

The malaria-causing Plasmodium parasites are transmitted to vertebrates by mosquitoes. To support their growth and replication, these intracellular parasites, which belong to the phylum Apicomplexa, have developed mechanisms to exploit their hosts. These mechanisms include expropriation of small metabolites from infected host cells, such as purine nucleotides and amino acids. Heretofore, no evidence suggested that transfer RNAs (tRNAs) could also be exploited. We identified an unusual gene in Apicomplexa with a coding sequence for membrane-docking and structure-specific tRNA binding. This Apicomplexa protein-designated tRip (tRNA import protein)-is anchored to the parasite plasma membrane and directs import of exogenous tRNAs. In the absence of tRip, the fitness of the parasite stage that multiplies in the blood is significantly reduced, indicating that the parasite may need host tRNAs to sustain its own translation and/or as regulatory RNAs. Plasmodium is thus the first example, to our knowledge, of a cell importing exogenous tRNAs, suggesting a remarkable adaptation of this parasite to extend its reach into host cell biology.

Isolated Intraorbital Frontosphenoidal Synostosis.

Unilateral anterior plagiocephaly is most commonly the result of deformational plagiocephaly or unilateral coronal synostosis, a premature fusion of the frontoparietal suture. However, other sutures within the coronal ring have been implicated in producing anterior cranial asymmetries. These fusions can occur in isolation or in concert with adjacent sutures. The frontosphenoidal suture is one such suture within the coronal ring that has been involved both concomitantly with and independently of frontoparietal suture fusion. Although isolated frontosphenoidal synostosis has been presented previously in the literature, these reports include patients with fusion of the extraorbital portion of the frontosphenoidal suture. This clinical report presents the first clearly documented patient of isolated frontosphenoidal synostosis that occurs entirely within the intraorbital region.The patient presented to Plastic Surgery Clinic at 3 months of age with left frontal flattening, supraorbital rim retrusion, and temporal bulging that was noted soon after birth. Computed tomography analysis revealed an isolated fusion of the greater and lesser wings of the sphenoid bone to the frontal bone on the left side. The patient had no family history of cranial anomalies and genetic testing was negative for mutations. The infant was treated with a cranial orthotic for 3 months, underwent open fronto-orbital advancement and cranial vault remodeling at 6 months, and continued wearing a cranial orthotic for another 4.5 months. Following surgical and orthotic treatment, the patient achieved a satisfactory result.

The Plasmodium translocon of exported proteins component EXP2 is critical for establishing a patent malaria infection in mice.

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium translocon of exported proteins (PTEX) and a cleavable Plasmodium export element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic Plasmodium berghei parasites deficient in EXP2, the putative pore-forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood-stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL-bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL-bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection.

Yeast lysates carrying the nucleoprotein from measles virus vaccine as a novel subunit vaccine platform to deliver Plasmodium circumsporozoite antigen.

BACKGROUND: Yeast cells represent an established bioreactor to produce recombinant proteins for subunit vaccine development. In addition, delivery of vaccine antigens directly within heat-inactivated yeast cells is attractive due to the adjuvancy provided by the yeast cell. In this study, Pichia pastoris yeast lysates carrying the nucleoprotein (N) from the measles vaccine virus were evaluated as a novel subunit vaccine platform to deliver the circumsporozoite surface antigen (CS) of Plasmodium. When expressed in Pichia pastoris yeast, the N protein auto-assembles into highly multimeric ribonucleoparticles (RNPs). The CS antigen from Plasmodium berghei (PbCS) was expressed in Pichia pastoris yeast in fusion with N, generating recombinant PbCS-carrying RNPs in the cytoplasm of yeast cells. RESULTS: When evaluated in mice after 3-5 weekly subcutaneous injections, yeast lysates containing N-PbCS RNPs elicited strong anti-PbCS humoral responses, which were PbCS-dose dependent and reached a plateau by the pre-challenge time point. Protective efficacy of yeast lysates was dose-dependent, although anti-PbCS antibody titers were not predictive of protection. Multimerization of PbCS on RNPs was essential for providing benefit against infection, as immunization with monomeric PbCS delivered in yeast lysates was not protective. Three weekly injections with N-PbCS yeast lysates in combination with alum adjuvant produced sterile protection in two out of six mice, and significantly reduced parasitaemia in the other individuals from the same group. This parasitaemia decrease was of the same extent as in mice immunized with non-adjuvanted N-PbCS yeast lysates, providing evidence that the yeast lysate formulation did not require accessory adjuvants for eliciting efficient parasitaemia reduction. CONCLUSIONS: This study demonstrates that yeast lysates are an attractive auto-adjuvant and efficient platform for delivering multimeric PbCS on measles N-based RNPs. By combining yeast lysates that carry RNPs with a large panel of Plasmodium antigens, this technology could be applied to developing a multivalent vaccine against malaria.

A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development.

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.

Plasmodium berghei histamine-releasing factor favours liver-stage development via inhibition of IL-6 production and associates with a severe outcome of disease.

Plasmodium spp., which causes malaria, produces a histamine-releasing factor (HRF), an orthologue of mammalian HRF. Histamine-releasing factor produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre-erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito-injected parasite form into the erythrocyte-infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL-6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti-IL-6 antibodies or infection in IL-6-deficient mice and parasite HRF is sufficient to decrease IL-6 synthesis, indicating a direct role of parasite HRF in reducing host IL-6. The target cells modulated by HRF for IL-6 production at early time points during liver infection are neutrophils. Parasite HRF is thus used to down-regulate a cytokine with anti-parasite activity. Our data also highlight the link between a prolonged transition from liver to blood-stage infection and reduced incidence of experimental cerebral malaria.

Local immune response to injection of Plasmodium sporozoites into the skin.

Malarial infection is initiated when the sporozoite form of the Plasmodium parasite is inoculated into the skin by a mosquito. Sporozoites invade hepatocytes in the liver and develop into the erythrocyte-infecting form of the parasite, the cause of clinical blood infection. Protection against parasite development in the liver can be induced by injection of live attenuated parasites that do not develop in the liver and thus do not cause blood infection. Radiation-attenuated sporozoites (RAS) and genetically attenuated parasites are now considered as lead candidates for vaccination of humans against malaria. Although the skin appears as the preferable administration route, most studies in rodents, which have served as model systems, have been performed after i.v. injection of attenuated sporozoites. In this study, we analyzed the early response to Plasmodium berghei RAS or wild-type sporozoites (WTS) injected intradermally into C57BL/6 mice. We show that RAS have a similar in vivo distribution to WTS and that both induce a similar inflammatory response consisting of a biphasic recruitment of polymorphonuclear neutrophils and inflammatory monocytes in the skin injection site and proximal draining lymph node (dLN). Both WTS and RAS associate with neutrophils and resident myeloid cells in the skin and the dLN, transform inside CD11b(+) cells, and induce a Th1 cytokine profile in the dLN. WTS and RAS are also similarly capable of priming parasite-specific CD8(+) T cells. These studies delineate the early and local response to sporozoite injection into the skin, and suggest that WTS and RAS prime the host immune system in a similar fashion.

In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages.

CD8(+) T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8(+) T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8(+) T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8(+) T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8(+) T cells and signaling by G protein-coupled receptors, although CD8(+) T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8(+) T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8(+) T cells.

Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity.

Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (Δhmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in Δhmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4(+) and CD8(+) T cells, including CD8(+) granzyme B(+) and CD8(+) IFN-γ(+) cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria.

Plasmodium and mononuclear phagocytes.

Plasmodium, the causative agent of malaria, initially multiplies inside liver cells and then in successive cycles inside erythrocytes, causing the symptoms of the disease. In this review, we discuss interactions between the extracellular and intracellular forms of the Plasmodium parasite and innate immune cells in the mammalian host, with a special emphasis on mononuclear phagocytes. We overview here what is known about the innate immune cells that interact with parasites, mechanisms used by the parasite to evade them, and the protective or detrimental contribution of these interactions on parasite progression through its life cycle and pathology in the host.

Calcium dynamics of Plasmodium berghei sporozoite motility.

Calcium is a key signalling molecule in apicomplexan parasites and plays an important role in diverse processes including gliding motility. Gliding is essential for the malaria parasite to migrate from the skin to the liver as well as to invade host tissues and cells. Here we investigated the dynamics of intracellular Ca(2+) in the motility of Plasmodium berghei sporozoites by live imaging and flow cytometry. We found that cytosolic levels of Ca(2+) increase when sporozoites are activated in suspension, which is sufficient to induce the secretion of integrin-like adhesins that are essential for gliding motility. By increasing intracellular Ca(2+) levels artificially with an ionophore, these adhesins are secreted onto the sporozoite surface, however, the parasite is not capable of gliding. A second level of Ca(2+) modulation was observed during attachment to and detachment from a solid substrate, leading to a further increase or a decrease in the cytoplasmic levels of Ca(2+) respectively. We also observed oscillations in the intracellular Ca(2+) level during gliding. Finally, an intracellular Ca(2+) chelator, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), and an inhibitor of the inositol triphosphate (IP3) receptor blocked the rise in intracellular Ca(2+) , adhesin secretion, and motility of activated sporozoites, indicating that intracellular stores supply Ca(2+) during sporozoite gliding. Our study indicates that a rise in intracellular Ca(2+) is necessary but not sufficient to activate gliding, that Ca(2+) levels are modulated in several ways during motility, and that a PI-PLC/IP3 pathway regulates Ca(2+) release during the process of sporozoite locomotion.

The role of MACPF proteins in the biology of malaria and other apicomplexan parasites.

Apicomplexans are eukaryotic parasites of major medical and veterinary importance. They have complex life cycles through frequently more than one host, interact with many cell types in their hosts, and can breach host cell membranes during parasite traversal of, or egress from, host cells. Some of these parasites make a strikingly heavy use of the pore-forming MACPF domain, and encode up to 10 different MACPF domain-containing proteins. In this chapter, we focus on the two most studied and medically important apicomplexans, Plasmodium and Toxoplasma, and describe the known functions of their MACPF polypeptide arsenal. Apicomplexan MACPF proteins appear to be involved in a variety of membrane-damaging events, making them an attractive model to dissect the structure-function relationships of the MACPF domain.

PK4, a eukaryotic initiation factor 2α(eIF2α) kinase, is essential for the development of the erythrocytic cycle of Plasmodium.

In response to environmental stresses, the mammalian serine threonine kinases PERK, GCN2, HRI, and PKR phosphorylate the regulatory serine 51 of the eukaryotic translation initiation factor 2α (eIF2α) to inhibit global protein synthesis. Plasmodium, the protozoan that causes malaria, expresses three eIF2α kinases: IK1, IK2, and PK4. Like GCN2, IK1 regulates stress response to amino acid starvation. IK2 inhibits development of malaria sporozoites present in the mosquito salivary glands. Here we show that the phosphorylation by PK4 of the regulatory serine 59 of Plasmodium eIF2α is essential for the completion of the parasite's erythrocytic cycle that causes disease in humans. PK4 activity leads to the arrest of global protein synthesis in schizonts, where ontogeny of daughter merozoites takes place, and in gametocytes that infect Anopheles mosquitoes. The implication of these findings is that drugs that reduce PK4 activity should alleviate disease and inhibit malaria transmission.

Development of Volunteer International Craniofacial Surgery Missions: The Komedyplast Protocol.

Volunteer surgical missions to provide cleft care to patients in developing countries has been done successfully for a number of years. Similar missions that provide craniofacial surgery introduce a dramatic step up in complexity. While articles have addressed protocols for the safe delivery of cleft care around the world, little has been written on volunteer craniofacial surgical missions. Komedyplast was established in March 2001 as a 501c(3) nonprofit organization to provide craniofacial surgical care to underserved populations and educate local surgeons in craniofacial principles. During 9 annual missions, the organization has provided surgical care to more than 150 patients with various complex, congenital, craniofacial conditions. The article addresses important safeguards that have been implemented to maximize safety and minimize risk.

A key role for Plasmodium subtilisin-like SUB1 protease in egress of malaria parasites from host hepatocytes.

In their mammalian host, Plasmodium parasites have two obligatory intracellular development phases, first in hepatocytes and subsequently in erythrocytes. Both involve an orchestrated process of invasion into and egress from host cells. The Plasmodium SUB1 protease plays a dual role at the blood stage by enabling egress of the progeny merozoites from the infected erythrocyte and priming merozoites for subsequent erythrocyte invasion. Here, using conditional mutagenesis in P. berghei, we show that SUB1 plays an essential role at the hepatic stage. Stage-specific sub1 invalidation during prehepatocytic development showed that SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and to egress from hepatocytes. Furthermore, mechanically released parasites were not adequately primed and failed to establish a blood stage infection in vivo. The critical involvement of SUB1 in both pre-erythrocytic and erythrocytic developmental phases qualifies SUB1 as an attractive multistage target for prophylactic and therapeutic anti-Plasmodium intervention strategies.

Liver-specific protein 2: a Plasmodium protein exported to the hepatocyte cytoplasm and required for merozoite formation.

The liver stage is the first stage of the malaria parasite that replicates in the vertebrate host. However, little is known about the interplay between the parasite liver stage and its host cell, the hepatocyte. In this study, we identified an exported protein that has a critical role in parasite development in host hepatocytes. Expressed sequence tag analysis of Plasmodium berghei liver-stage parasites indicated that transcripts encoding a protein with an N-terminal signal peptide, designated liver-specific protein 2 (LISP2), are highly expressed in this stage. Expression of LISP2 was first observed 24 h after infection and rapidly increased during the liver-stage schizogony. Immunofluorescent staining with anti-LSP2 antibodies showed that LISP2 was carried to the parasitophorous vacuole and subsequently transported to the cytoplasm and nucleus of host hepatocytes. Gene targeting experiments demonstrated that majority of the LISP2-mutant liver-stage parasites arrested their development during formation of merozoites. These results indicate that exported LISP2 is involved in parasite-host interactions required for the development of liver-stage parasites inside hepatocytes. This study demonstrated that mid-to-late liver-stage malarial parasites have a system for exporting proteins to the host cell as intraerythrocytic stages do and presumably to use the proteins to modify the host cell and improve the environment.

Small-molecule inhibitors of the pseudaminic acid biosynthetic pathway: targeting motility as a key bacterial virulence factor.

Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 µM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 µM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 µM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.