A role for LHC1 in higher order structure and complement binding of the Cryptococcus neoformans capsule.

Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.

Is Botulinum Toxin Type A a Valuable Adjunct During Femoral Lengthening? A Randomized Trial.

BACKGROUND: Reduced joint ROM and distraction-induced pain are common complaints of patients who have undergone gradual femoral lengthening. Attempts to reduce the effects of lengthening on joint motion have included the use of botulinum toxin to reduce the muscle forces that restrict motion. The benefits of this approach during femoral lengthening, however, have not been conclusively established. QUESTIONS/PURPOSES: We wished to evaluate the effects of botulinum toxin type A (BtX-A) injection in the anterior thigh muscles during femoral distraction osteogenesis on adjacent joint ROM and distraction-induced pain. We asked: (1) Does injection of BtX-A in the quadriceps muscles lead to improved knee and hip motion during femoral lengthening? (2) Does injection of BtX-A reduce pain during femoral lengthening? METHODS: A single-center, double-blind, randomized placebo-controlled trial was conducted. Forty-four patients (88 femurs) undergoing bilateral femoral lengthening for familial short stature were included in the study. BtX-A (200 IU) was injected intraoperatively in the quadriceps muscles of one thigh. An equal volume of sterile normal saline was injected in the other thigh as a control. Selection of the limb receiving the toxin was randomized. Clinical evaluation included a VAS score for pain measurement, ROM evaluation of the hips and knees, and measurement of thigh circumference. Side-to-side differences were analyzed throughout the entire consolidation phase. No patients were lost to followup, leaving 44 patients (88 femurs). The mean followup was 26 months (range, 14-40 months). The distraction rate and final length of gain were similar between treated and control limbs. A priori power analysis suggested that 44 legs were required in each group to achieve statistical significance of 0.05 with 90% power to detect a 50% difference in treatment effect between treatment and control groups. RESULTS: There were no differences in hip ROM, knee ROM, or maximal thigh circumference between the two lower extremities at any time during the study period. VAS scores were no different between the patients who received BtX-A and those who received saline. CONCLUSIONS: Local injection of 200 IU BtX-A in the quadriceps muscles does not appear to reduce distraction-induced pain nor enhance ROM in the hip or knee during femoral lengthening. Additional studies are needed to evaluate the effect of larger doses or different injection methods. Based on our findings, we do not recommend routine use of botulinum injections during limb lengthening and believe any further use of this drug should only be in the context of a controlled trial. LEVEL OF EVIDENCE: Level II, therapeutic study.

Fixator-assisted Technique Enables Less Invasive Plate Osteosynthesis in Medial Opening-wedge High Tibial Osteotomy: A Novel Technique.

BACKGROUND: Opening-wedge high tibial osteotomy is a well-established procedure in the management of medial osteoarthritis of the knee and correction of proximal tibia vara. Recently, surgical approaches using less invasive plate osteosynthesis have been used with the goal of minimizing complications from more extensive soft tissue exposures. However, to our knowledge, less invasive fixator-assisted plate osteosynthesis has not been tested in the setting of opening-wedge high tibial osteotomy. QUESTIONS/PURPOSES: The purposes of this study were (1) to assess the complications associated with use of a fixator-assisted less invasive plate osteosynthesis technique to stabilize an opening-wedge high tibial osteotomy in the treatment of proximal tibial vara; and (2) to evaluate the ability of this technique to achieve correction of the proximal tibial deformity and achieve osseous union. METHODS: From June 2011 to June 2013, a total of 157 limbs in 83 patients who underwent fixator-assisted high tibial osteotomy for (1) idiopathic genu vara; or (2) osteoarthritis of the knee with proximal tibia vara were initially enrolled. Of these, eight limbs (5%) were excluded on the way; thus, 149 limbs in 77 patients were evaluated. During the period in question, no other techniques were used for proximal tibial osteotomy. The surgical procedures included less preparation of soft tissue, proximal tibial osteotomy, application of a temporary external fixator, correction of alignment, and final fixation with the help of an external fixator. Complications were assessed by chart review and the alignment in both coronal and sagittal planes was compared pre- and postoperatively. Radiographic review to confirm osseous union and alignment was performed by two of the authors not involved in clinical care of the patient. Delayed union was described as union occurring later than 4 months. RESULTS: Thirty limbs out of 149 tibiae (20%) showed complications, all of which were resolved without leaving any sequela. Twenty-seven limbs out of 149 limbs (18%) showed lateral cortical hinge fracture and three limbs out of 149 limbs (2%) showed soft tissue complications (two superficial infections, one wound hematoma). The overall completeness of reaching the target correction was excellent. In the coronal plane, the difference between the amount of real correction and the amount of target correction was 0.3° ± 0.7° (p < 0.001). In the sagittal plane, the difference between pre- and postoperative posterior proximal tibial angle was -0.1° ± 0.2° (p < 0.001). All osteotomies healed before 4 months. CONCLUSIONS: Fixator-assisted high tibial osteotomy is a valid option for medial opening-wedge high tibial osteotomy, which enables less invasive surgery with excellent coronal/sagittal/rotational alignment control. However, future studies should compare this approach with other approaches for proximal tibial osteotomy to ascertain whether indeed this procedure is less invasive or more reliable. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Mating pheromone in Cryptococcus neoformans is regulated by a transcriptional/degradative "futile" cycle.

Sexual reproduction in fungi requires induction of signaling pheromones within environments that are conducive to mating. The fungus Cryptococcus neoformans is currently the fourth greatest cause of infectious death in regions of Africa and undergoes mating in phytonutrient-rich environments to create spores with infectious potential. Here we show that under conditions where sexual development is inhibited, a ∼17-fold excess of MFα pheromone transcript is synthesized and then degraded by a DEAD box protein, Vad1, resulting in low steady state transcript levels. Transfer to mating medium or deletion of the VAD1 gene resulted in high level accumulation of MFα transcripts and enhanced mating, acting in concert with the mating-related HOG1 pathway. We then investigated whether the high metabolic cost of this apparently futile transcriptional cycle could be justified by a more rapid induction of mating. Maintenance of Vad1 activity on inductive mating medium by constitutive expression resulted in repressed levels of MFα that did not prevent but rather prolonged the time to successful mating from 5-6 h to 15 h (p < 0.0001). In sum, these data suggest that VAD1 negatively regulates the sexual cell cycle via degradation of constitutive high levels of MFα transcripts in a synthetic/degradative cycle, providing a mechanism of mRNA induction for time-critical cellular events, such as mating induction.

PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans.

Autophagy is a process by which cells recycle cytoplasm and defective organelles during stress situations such as nutrient starvation. It can also be used by host cells as an immune defense mechanism to eliminate infectious pathogens. Here we describe the use of autophagy as a survival mechanism and virulence-associated trait by the human fungal pathogen Cryptococcus neoformans. We report that a mutant form of C. neoformans lacking the Vps34 PI3K (vps34Delta), which is known to be involved in autophagy in ascomycete yeast, was defective in the formation of autophagy-related 8-labeled (Atg8-labeled) vesicles and showed a dramatic attenuation in virulence in mouse models of infection. In addition, autophagic vesicles were observed in WT but not vps34Delta cells after phagocytosis by a murine macrophage cell line, and Atg8 expression was exhibited in WT C. neoformans during human infection of brain. To dissect the contribution of defective autophagy in vps34Delta C. neoformans during pathogenesis, a strain of C. neoformans in which Atg8 expression was knocked down by RNA interference was constructed and these fungi also demonstrated markedly attenuated virulence in a mouse model of infection. These results demonstrated PI3K signaling and autophagy as a virulence-associated trait and survival mechanism during infection with a fungal pathogen. Moreover, the data show that molecular dissection of such pathogen stress-response pathways may identify new approaches for chemotherapeutic interventions.

Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans.

The study of regulatory networks in human pathogens such as Cryptococcus neoformans provides insights into host-pathogen interactions that may allow for correlation of gene expression patterns with clinical outcomes. In the present study, deletion of the cryptococcal copper-dependent transcription factor 1 (Cuf1) led to defects in growth and virulence factor expression in low copper conditions. In mouse models, cuf1Delta strains exhibited reduced dissemination to the brain, but no change in lung growth, suggesting copper is limiting in neurologic infections. To examine this further, a biologic probe of available copper was constructed using the cryptococcal CUF1-dependent copper transporter, CTR4. Fungal cells demonstrated high CTR4 expression levels after phagocytosis by macrophage-like J774.16 cells and during infection of mouse brains, but not lungs, consistent with limited copper availability during neurologic infection. This was extended to human brain infections by demonstrating CTR4 expression during C. neoformans infection of an AIDS patient. Moreover, high CTR4 expression by cryptococcal strains from 24 solid organ transplant patients was associated with dissemination to the CNS. Our results suggest that copper acquisition plays a central role in fungal pathogenesis during neurologic infection and that measurement of stable traits such as CTR4 expression may be useful for risk stratification of individuals with cryptococcosis.

Overexpression of TUF1 restores respiratory growth and fluconazole sensitivity to a Cryptococcus neoformans vad1Delta mutant.

The yeast-like fungus Cryptococcus neoformans favours respiration as a mechanism of energy production, and thus depends heavily on mitochondrial function. Previous studies of a C. neoformans vad1Delta mutant revealed reduced expression of the mitochondrial elongation factor TUF1 and defects in glycerol utilization, consistent with mitochondrial dysfunction. In this study, we found that in trans expression of TUF1 in the vad1Delta mutant suppressed the mitochondrial defects, including growth on respiration-dependent carbon sources and fluconazole resistance, associated with VAD1 deletion. Tetracycline, an inhibitor of mitochondrial translation, was found to confer resistance to fluconazole in the wild-type and vad1Delta mutant, whereas the fluconazole susceptibility of the TUF1-overexpressing strain was unaffected by tetracycline treatment. In the presence of fluconazole, the vad1Delta mutant exhibited increased activation of the global transcriptional regulator Sre1. TUF1 overexpression failed to alter cleavage of Sre1 in response to fluconazole in the vad1Delta mutant, suggesting that TUF1 repression in the vad1Delta mutant is distal to Sre1, or that it occurs through an independent pathway.

CD14-dependent modulation of NF-kappaB alternative splicing in the lung after burn injury.

Nuclear factor kappa-B (NF-kappaB), a key downstream player of the LPS signaling pathway, has been shown to undergo alternative splicing in in vitro studies. In this study, we examined the effect of injury and the role of CD14 on NF-kappaB alternative splicing using a murine burn model. CD14 knockout and respective wild-type mice were sacrificed after 18% total body surface area burn. RT-PCR and subsequent sequencing analysis revealed that injury induced multiple novel splicing variants of relA, relB, and NF-kappaB2 in the lungs of CD14 knockout but not wild-type mice. These novel variants resulted either from exon skipping, alternative usage of splicing signals, or intron retention. All but one variant resulted in a frameshift leading to premature termination of translation. These splicing variants encoded for proteins that lacked the domains essential for NF-kappaB transcription factor functions. Two NF-kappaB2 variants acquired only minor changes in their C-terminus that might affect their post-translational cleavage into active isoforms. These results suggest that alternative splicing may be one of the mechanisms by which NF-kappaB activity in the lungs can be regulated after injury. Furthermore, the CD14-mediated LPS signaling pathway may play a role in the regulation of NF-kappaB alternative splicing in the lungs after injury.

Downregulation of NF-kappaB activity associated with alteration in proliferative response in the spleen after burn injury.

Alterations in proliferation status and cellular composition of immune organs are among key events in the modulation of immune function after burn injury. Nuclear factor (NF)-kappaB is a transcription factor that plays a pivotal role in the response to injury as well as immune cell differentiation and proliferation. In this study, we investigated the effects of burn injury on the activity of NF-kappaB and its association with cellular proliferation in the spleen. Western analysis of whole spleen tissues of mice after 18% burn injury revealed a marked reduction in nuclear NF-kappaB rel A protein expression 3 to 21 days after injury when there was an increase in proliferative activity in the red pulp of the spleen after injury as indicated by an increase in proliferating cell nuclear antigen (PCNA). In the splenic B cells, however, the down-regulation of NF-kappaB rel A was associated with decreased PCNA expression as well as IkappaBalpha and phosphorylated IkappaBalpha. In contrast, no significant change in NF-kappaB rel A or PCNA expression was observed for splenic T cells. These data suggest that there is a differential regulation of NF-kappaB and proliferative activity in the splenic cell subsets after burn injury. Furthermore, the regulation of NF-kappaB may be linked to the proliferative changes seen in the spleen after burn injury.

Binding of serum mannan binding lectin to a cell integrity-defective Cryptococcus neoformans ccr4Delta mutant.

Mannan binding lectin (MBL) is an innate immune mediator belonging to the collectin family known to bind to the surfaces of many viruses, bacteria, and fungi. However, pathogenic strains of the fungus Cryptococcus neoformans are resistant to MBL binding. To dissect the mechanism of cryptococcal resistance to MBL, we compared MBL binding to an encapsulated wild-type strain, an encapsulated ccr4Delta mutant defective in cell integrity, and an acapsular cap60Delta strain. No MBL binding was detected on wild-type C. neoformans. In contrast, the ccr4Delta mutant bound MBL to the cell wall, predominantly at the ends of enlarged buds, whereas the acapsular strain bound MBL only at the bud neck and bud scars. In addition, the ccr4Delta mutant was sensitive to the cell wall-active antifungal caspofungin and other cell wall stress inducers, and its virulence was reduced in a mouse model of cryptococcosis. Interestingly, treatment of wild-type cells with caspofungin also increased MBL binding to C. neoformans. These results suggest that both the presence of capsule and wild-type cell wall architecture preclude MBL binding to C. neoformans.

Cell wall targeting of laccase of Cryptococcus neoformans during infection of mice.

Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1Delta cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.

The Hsp70 member, Ssa1, acts as a DNA-binding transcriptional co-activator of laccase in Cryptococcus neoformans.

Hsp70 proteins are a well-known class of chaperones that have also been described to have roles in cellular regulation. Here, we show that a Cryptococcus neoformans Hsp70 homologue Ssa1 acts as a DNA-binding transcriptional co-activator of the fungal virulence factor, laccase, via binding to a GC-rich element within the 5'-UAS in response to glucose starvation, iron, copper, calcium and temperature. In addition, Ssa1 forms a regulatory complex with heat shock transcription factor and TATA-binding protein during laccase induction. Furthermore, deletion of Ssa1 results in reduced laccase and attenuated virulence using a mouse model. These results indicate that Hsp70 functions as a stress-related transcriptional co-activator required for fungal virulence.