Publisher Correction: Complement-dependent outer membrane perturbation sensitizes Gram-negative bacteria to Gram-positive specific antibiotics.

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Complement-dependent outer membrane perturbation sensitizes Gram-negative bacteria to Gram-positive specific antibiotics.

Gram-negative bacteria are refractory to the action of many antibiotics due to their impermeable outer membrane. An important player of the immune system is the complement system, a protein network in serum that directly kills Gram-negative bacteria through pore-formation by the Membrane Attack Complexes (MAC). We here show that the MAC rapidly perforates the outer membrane but that inner membrane damage, which is essential for killing, is relatively slow. Importantly, we demonstrate that MAC-induced outer membrane damage sensitizes Gram-negative bacteria to otherwise ineffective, Gram-positive-specific, antimicrobials. Synergy between serum and nisin was observed for 22 out of 53 tested Gram-negative clinical isolates and for multi-drug resistant (MDR) blood isolates. The in vivo relevance of this process is further highlighted by the fact that blood sensitizes a MDR K. pneumoniae strain to vancomycin. Altogether, these data imply that antibiotics that are considered ineffective to treat infections with Gram-negatives may have different functional outcomes in patients, due to the presence of the complement system.

Staphylococcus aureus protects its immune-evasion proteins against degradation by neutrophil serine proteases.

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.

Anti-opsonic properties of staphylokinase.

Recently we described a novel bacteriophage-encoded pathogenicity island in Staphylococcus aureus that harbors a number of virulence factors that are all involved in the evasion of innate immunity. Here we describe a mechanism by which staphylokinase (SAK), frequently present on this pathogenicity island, interferes with innate immune defenses: SAK is anti-opsonic. By activating human plasminogen (PLG) into plasmin (PL) at the bacterial surface, it creates bacterium-bound serine protease activity that leads to degradation of two major opsonins: human immunoglobulin G (IgG) and human C3b. Incubation of opsonized bacteria with PLG and SAK resulted in removal of anti-staphylococcal IgGs and C3b from the bacterial surface. In phagocytosis assays this proved to be a very efficient mechanism to reduce the opsonic activity of human IgG and serum. The fact that SAK activates human PLG at the bacterial surface and removes IgG as well as C3b makes this protein a unique anti-opsonic molecule.

Antibody neutralization of vascular endothelial growth factor (VEGF) fails to attenuate vascular permeability and brain edema in experimental pneumococcal meningitis.

To determine the contribution of vascular endothelial growth factor (VEGF) to cerebral edema formation in bacterial meningitis, we used a VEGF neutralizing antibody to block VEGF in rabbits, following induction of meningitis by intracisternal inoculation with 10(9) heat-killed pneumococci. At 8 h, cerebrospinal fluid (CSF) VEGF was significantly elevated in infected untreated animals, and correlated with CSF white blood cell (WBC) count (r=0.56, P=0.004), and brain water content (r=0.42, P=0.04). Blocking of VEGF did not attenuate brain edema, blood-brain barrier disruption, or CSF pleocytosis. The functional role of VEGF in the pathophysiology of BM remains elusive.

A new approach to measure single-event related brain activity using real-time fMRI: feasibility of sensory, motor, and higher cognitive tasks.

Real-time fMRI is a rapidly emerging methodology that enables monitoring changes in brain activity during an ongoing experiment. In this article we demonstrate the feasibility of performing single-event sensory, motor, and higher cognitive tasks in real-time on a clinical whole-body scanner. This approach requires sensitivity optimized fMRI methods: Using statistical parametric mapping we quantified the spatial extent of BOLD contrast signal changes as a function of voxel size and demonstrate that sacrificing spatial resolution and readout bandwidth improves the detection of signal changes in real time. Further increases in BOLD contrast sensitivity were obtained by using real-time multi-echo EPI. Real-time image analysis was performed using our previously described Functional Imaging in REal time (FIRE) software package, which features real-time motion compensation, sliding window correlation analysis, and automatic reference vector optimization. This new fMRI methodology was validated using single-block design paradigms of standard visual, motor, and auditory tasks. Further, we demonstrate the sensitivity of this method for online detection of higher cognitive functions during a language task using single-block design paradigms. Finally, we used single-event fMRI to characterize the variability of the hemodynamic impulse response in primary and supplementary motor cortex in consecutive trials using single movements. Real-time fMRI can improve reliability of clinical and research studies and offers new opportunities for studying higher cognitive functions.

The effect of sequence repeat time on auditory cortex stimulation during phonetic discrimination.

Acoustic noise generated by the MR scanner gradient system during fMRI studies of auditory function is a very significant potential confound. Despite these deleterious effects, fMRI of the auditory cortex has been successful and numerous practitioners have circumvented the problem of acoustic masking noise. In the context of auditory cortex fMRI, the sequence repeat time (TR) has the effect of altering the length of time during which the scanner is quiet. Indeed, the move to whole-brain fMRI makes the problem of acoustic noise more acute and points to the need to examine the role of TR and its influence on the BOLD signal. The aim of this study was to examine the effect of varying the TR time on activation of auditory cortex during presentation and performance of a phonetic discrimination task. The results presented here demonstrate that the influence of sequence repeat time is considerable. For a short repeat time it is likely that the noise from the scanner is a significant mask and hinders accurate task performance. At the other extreme, a repeat time of 9 s is also suboptimal, probably due to attentional effects and lack of concentration and not least because of the longer overall measurement times. The results of this study point to a complicated interplay between psychophysical factors as well as physical parameters; attention, acoustic noise, total duration of the experiment, consideration of the volume of acquisition, and overall difficulty of the task have to be assessed and balanced. For the paradigm used here, the results suggest an optimal TR of around 6 s for a 16-slice acquisition.

Line bisection judgments implicate right parietal cortex and cerebellum as assessed by fMRI.

OBJECTIVE: To use functional MRI (fMRI) to determine which brain regions are implicated when normal volunteers judge whether pretransected horizontal lines are correctly bisected (the Landmark test). BACKGROUND: Manual line bisection and a variant thereof involving perceptual judgments of pretransected lines (the Landmark test) are widely used to assess unilateral visuospatial neglect in patients with neurologic disease. Although unilateral (left) neglect most often results from lesions to right temporoparietal cortex, the normal functional anatomy of the Landmark test has not been convincingly demonstrated. METHODS: fMRI was carried out in 12 healthy right-handed male volunteers who judged whether horizontal lines were correctly prebisected. In the control task, subjects detected whether the horizontal lines contained a transection mark irrespective of the position of that mark. Response was by two-choice key press: on half the trials, subjects used the right, and on half, the left hand. Statistical analysis of evoked blood oxygenation level-dependent responses, measured with echoplanar imaging, employed statistical parametric mapping. RESULTS: Performing the Landmark task showed neural activity (p < 0.05, corrected) in the right superior posterior and right inferior parietal lobe, early visual processing areas bilaterally, the cerebellar vermis, and the left cerebellar hemisphere. Only the latter area showed a significant interaction with hand used. CONCLUSIONS: The right hemispheric dominance observed in inferior parietal cortex is consistent with the results of lesion studies. Right superior parietal cortex, vermis, and left cerebellar hemisphere have not been implicated in neglect, but all appear to play a cognitive role in the Landmark task.

The network of brain areas involved in the motion aftereffect.

A network of brain areas is expected to be involved in supporting the motion aftereffect. The most active components of this network were determined by means of an fMRI study of nine subjects exposed to a visual stimulus of moving bars producing the effect. Across the subjects, common areas were identified during various stages of the effect, as well as networks of areas specific to a single stage. In addition to the well-known motion-sensitive area MT the prefrontal brain areas BA44 and 47 and the cingulate gyrus, as well as posterior sites such as BA37 and BA40, were important components during the period of the motion aftereffect experience. They appear to be involved in control circuitry for selecting which of a number of processing styles is appropriate. The experimental fMRI results of the activation levels and their time courses for the various areas are explored. Correlation analysis shows that there are effectively two separate and weakly coupled networks involved in the total process. Implications of the results for awareness of the effect itself are briefly considered in the final discussion.

Enhancement of BOLD-contrast sensitivity by single-shot multi-echo functional MR imaging.

Improved data acquisition and processing strategies for blood oxygenation level-dependent (BOLD)-contrast functional magnetic resonance imaging (fMRI), which enhance the functional contrast-to-noise ratio (CNR) by sampling multiple echo times in a single shot, are described. The dependence of the CNR on T2*, the image encoding time, and the number of sampled echo times are investigated for exponential fitting, echo summation, weighted echo summation, and averaging of correlation maps obtained at different echo times. The method is validated in vivo using visual stimulation and turbo proton echoplanar spectroscopic imaging (turbo-PEPSI), a new single-shot multi-slice MR spectroscopic imaging technique, which acquires up to 12 consecutive echoplanar images with echo times ranging from 12 to 213 msec. Quantitative T2*-mapping significantly increases the measured extent of activation and the mean correlation coefficient compared with conventional echoplanar imaging. The sensitivity gain with echo summation, which is computationally efficient provides similar sensitivity as fitting. For all data processing methods sensitivity is optimum when echo times up to 3.2 T2* are sampled. This methodology has implications for comparing functional sensitivity at different magnetic field strengths and between brain regions with different magnetic field inhomogeneities.

Influence of acoustic masking noise in fMRI of the auditory cortex during phonetic discrimination.

The application of functional magnetic resonance imaging (fMRI) to study activation of auditory cortex suffers from one significant confounding factor, namely, that of the acoustic noise generated by the gradient system, which is an integral part of the imaging process. Earlier work has shown that it is indeed possible to distinguish cortical activation resulting from presentation of auditory stimuli despite the presence of background noise from the gradient system. The influence of acoustic noise from the gradient system of the MRI scanner on the blood oxygen level-dependent (BOLD) response during functional activation of the auditory cortex has been investigated in six healthy subjects with no hearing difficulties. Experiments were performed using gradient-echo echoplanar imaging (EPI) and a verbal, auditory discrimination paradigm, presented in a block-wise manner, in which carefully aligned consonant-vowel syllables were presented at a rate of 1 Hz. For each volunteer the experiment was repeated three times with all parameters fixed, except slice number, which was 4, 16, or 64. The positioning of the central four slices in each experiment was common. Thus, the fraction of TR during which the stimulus is on but no imaging is being performed, varies from almost zero, in the case of 64 slices, to over 8 seconds, in the case of four slices. Only the central four slices were of interest; additional slices simply generated acoustic noise and were discarded. During the four-slice experiment, all subjects showed a robust BOLD response in the superior temporal gyrus covering the primary and secondary auditory cortex. The spatial extent and the z-scores of the activated regions decreased with longer duration of gradient noise from the scanner. For a phonetic discrimination task, the results indicate that presentation of the stimulus during periods free from scanner noise leads to a more pronounced BOLD response.